Abstract:

Human antibodies, preferably recombinant human antibodies, that
specifically bind to human interleukin-12 (hIL-12) are disclosed.
Preferred antibodies have high affinity for hIL-12 and neutralize hIL-12
activity in vitro and in vivo. An antibody of the invention can be a
full-length antibody or an antigen-binding portion thereof. The
antibodies, or antibody portions, of the invention are useful for
detecting hIL-12 and for inhibiting hIL-12 activity, e.g., in a human
subject suffering from a disorder in which hIL-12 activity is
detrimental. Nucleic acids, vectors and host cells for expressing the
recombinant human antibodies of the invention, and methods of
synthesizing the recombinant human antibodies, are also encompassed by
the invention.

Claims:

1. An isolated antibody, or antigen binding portion thereof, that is
capable of binding to the p40 subunit of IL-12 and is capable of altering
the conformational structure of said p40 subunit of IL-12.

2. The isolated antibody of claim 1, or antigen binding portion thereof,
which dissociates from the p40 subunit of human IL-12 with a Kd of
1.times.10.sup.-10 M or less or a koff rate constant of
1.times.10.sup.-3 s-1 or less, as determined by surface plasmon
resonance.

3. The isolated antibody of claim 1, or antigen binding portion thereof,
which is a neutralizing antibody

4. The isolated antibody of claim 3, or antigen binding portion thereof,
which inhibits phytohemagglutinin blast proliferation in an in vitro PHA
assay with an IC50 of 1.times.10.sup.-9 M or less, or which inhibits
human IFNγ production with an IC50 of 1.times.10.sup.-10 M or
less.

5. The isolated antibody of claim 1, or antigen binding portion thereof,
which is a human antibody.

6. A pharmaceutical composition comprising the antibody or an antigen
binding portion thereof of claim 1, and a pharmaceutically acceptable
carrier.

7. A method for detecting the p40 subunit of an interleukin comprising
contacting the p40 subunit of IL-12 with the antibody, or antigen-binding
portion thereof, of claim 1 such that said p40 subunit is detected.

8. A method for detecting the p40 subunit of an interleukin comprising
contacting the p40 subunit of the interleukin with the antibody, or
antigen-binding portion thereof, of claim 1 such that the p40 subunit of
the interleukin is detected

9. The method of claim 7, wherein the p40 subunit of the interleukin is
detected in vitro.

10. The method of claim 7, wherein the p40 subunit of the interleukin is
detected in a biological sample for diagnostic purposes.

11. A method for inhibiting an activity of an interleukin comprising a p40
subunit, comprising contacting the interleukin with the antibody, or
antigen-binding portion thereof, of claim 1 such that the activity is
inhibited.

12. A method for inhibiting an activity of an interleukin comprising a p40
subunit in a human subject suffering from a disorder in which the
activity is detrimental, comprising administering to the human subject
the antibody, or antigen-binding portion thereof, of claim 1 such that
the activity in the human subject is inhibited.

13. The method of claim 11, wherein said interleukin is IL-12.

14. The method of claim 11, wherein said interleukin comprises a p40
subunit and a p19 subunit.

18. The method of claim 12, wherein the disorder is rheumatoid arthritis.

19. A method for altering the conformational structure of the p40 subunit
of IL-12, the method comprising contacting said subunit with an antibody,
or antigen binding portion thereof, that is capable of binding to said
subunit and is capable of altering the conformational structure of said
subunit in an amount effective to alter the conformational structure of
said subunit, thereby altering the conformational structure of said
subunit.

20. A method for inhibiting the activity of an interleukin comprising a
p40 subunit, the method comprising contacting the interleukin with an
antibody, or antigen binding portion thereof, that is capable of binding
to the p40 subunit of IL-12 and is capable of altering the conformational
structure of the interleukin in an amount effective to alter the
conformational structure of the interleukin, thereby inhibiting the
activity of the interleukin.

Description:

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/534,717, filed Mar. 24, 2000, which issued on
Jul. 5, 2005 as U.S. Pat. No. 6,914,128. This application also claims
priority to U.S. provisional application Ser. No. 60/126,603, filed Mar.
25, 1999. The entire contents of each of the foregoing applications are
hereby incorporated herein by reference.

[0003] Structurally, IL-12 is a heterodimeric protein comprising a 35 kDa
subunit (p35) and a 40 kDa subunit (p40) which are both linked together
by a disulfide bridge (referred to as the "p70 subunit"). The
heterodimeric protein is produced primarily by antigen-presenting cells
such as monocytes, macrophages and dendritic cells. These cell types also
secrete an excess of the p40 subunit relative to p70 subunit. The p40 and
p35 subunits are genetically unrelated and neither has been reported to
possess biological activity, although the p40 homodimer may function as
an IL-12 antagonist.

[0004] Functionally, IL-12 plays a central role in regulating the balance
between antigen specific T helper type (Th1) and type 2 (Th2)
lymphocytes. The Th1 and Th2 cells govern the initiation and progression
of autoimmune disorders, and IL-12 is critical in the regulation of
Th1-lymphocyte differentiation and maturation. Cytokines released by
the Th1 cells are inflammatory and include interferon γ
(IFNγ), IL-2 and lymphotoxin (LT). Th2 cells secrete IL-4, IL-5,
IL-6, IL-10 and IL-13 to facilitate humoral immunity, allergic reactions,
and immunosuppression.

[0005] Consistent with the preponderance of Th1 responses in autoimmune
diseases and the proinflammatory activities of IFNγ, IL-12 may play
a major role in the pathology associated with many autoimmune and
inflammatory diseases such as rheumatoid arthritis (RA), multiple
sclerosis (MS), and Crohn's disease.

[0008] Due to the role of human IL-12 in a variety of human disorders,
therapeutic strategies have been designed to inhibit or counteract IL-12
activity. In particular, antibodies that bind to, and neutralize, IL-12
have been sought as a means to inhibit IL-12 activity. Some of the
earliest antibodies were murine monoclonal antibodies (mAbs), secreted by
hybridomas prepared from lymphocytes of mice immunized with IL-12 (see
e.g., World Patent Application Publication No. WO 97/15327 by Strober et
al.; Neurath et al. (1995) J. Exp. Med. 182:1281-1290; Duchmann et al.
(1996) J. Immunol. 26:934-938). These murine IL-12 antibodies are limited
for their use in vivo due to problems associated with administration of
mouse antibodies to humans, such as short serum half life, an inability
to trigger certain human effector functions and elicitation of an
unwanted immune response against the mouse antibody in a human (the
"human anti-mouse antibody" (HAMA) reaction).

[0009] In general, attempts to overcome the problems associated with use
of fully-murine antibodies in humans, have involved genetically
engineering the antibodies to be more "human-like." For example, chimeric
antibodies, in which the variable regions of the antibody chains are
murine-derived and the constant regions of the antibody chains are
human-derived, have been prepared (Junghans, et al. (1990) Cancer Res.
50:1495-1502; Brown et al. (1991) Proc. Natl. Acad. Sci. 88:2663-2667;
Kettleborough et al. (1991) Protein Engineering. 4:773-783). However,
because these chimeric and humanized antibodies still retain some murine
sequences, they still may elicit an unwanted immune reaction, the human
anti-chimeric antibody (HACA) reaction, especially when administered for
prolonged periods.

[0010] A preferred IL-12 inhibitory agent to murine antibodies or
derivatives thereof (e.g., chimeric or humanized antibodies) would be an
entirely human anti-IL-12 antibody, since such an agent should not elicit
the HAMA reaction, even if used for prolonged periods. However, such
antibodies have not been described in the art and, therefore are still
needed.

SUMMARY OF THE INVENTION

[0011] The present invention provides human antibodies that bind human
IL-12. The invention also relates to the treatment or prevention of acute
or chronic diseases or conditions whose pathology involves IL-12, using
the human anti-IL-12 antibodies of the invention.

[0012] In one aspect, the invention provides an isolated human antibody,
or an antigen-binding portion thereof, that binds to human IL-12.

[0014] a human antibody or antigen-binding portion thereof, selectively
mutated at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid residue such
that it binds to human IL-12.

[0016] a human antibody or antigen-binding portion thereof, selectively
mutated at a preferred selective mutagenesis position with an activity
enhancing amino acid residue such that it binds to human IL-12.

[0017] In another preferred embodiment, the selectively mutated human
IL-12 antibody or antigen-binding portion thereof is selectively mutated
at more than one preferred selective mutagenesis position, contact or
hypermutation positions with an activity enhancing amino acid residue. In
another preferred embodiment, the selectively mutated human IL-12
antibody or antigen-binding portion thereof is selectively mutated at no
more than three preferred selective mutagenesis positions, contact or
hypermutation positions. In another preferred embodiment, the selectively
mutated human IL-12 antibody or antigen-binding portion thereof is
selectively mutated at no more than two preferred selective mutagenesis
position, contact or hypermutation positions. In yet another preferred
embodiment, the selectively mutated human IL-12 antibody or
antigen-binding portion thereof, is selectively mutated such that a
target specificity affinity level is attained, the target level being
improved over that attainable when selecting for an antibody against the
same antigen using phage display technology. In another preferred
embodiment, the selectively mutated human IL-12 antibody further retains
at least one desirable property or characteristic, e.g., preservation of
non-cross reactivity with other proteins or human tissues, preservation
of epitope recognition, production of an antibody with a close to a
germline immunoglobulin sequence.

[0018] In another embodiment, the invention provides an isolated human
antibody, or antigen-binding portion thereof, that binds to human IL-12
and dissociates from human IL-12 with a Koff rate constant of 0.1
s-1 or less, as determined by surface plasmon resonance, or which
inhibits phytohemagglutinin blast proliferation in an in vitro
phytohemagglutinin blast proliferation assay (PHA assay) with an
IC50 of 1×10-6 M or less. More preferably, the isolated
human antibody or an antigen-binding portion thereof, dissociates from
human IL-12 with a Koff rate constant of 1×10-2 s-1
or less, or inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC50 of 1×10-7 M or less. More
preferably, the isolated human antibody, or an antigen-binding portion
thereof, dissociates from human IL-12 with a Koff rate constant of
1×10-3 s-1 or less, or inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC50 of
1×10-8 M or less. More preferably, the isolated human
antibody, or an antigen-binding portion thereof, dissociates from human
IL-12 with a Koff rate constant of 1×10-4 s-1 or
less, or inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less. More
preferably, the isolated human antibody, or an antigen-binding portion
thereof, dissociates from human IL-12 with a Koff rate constant of
1×10-5 s-1 or less, or inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC50 of
1×10-10 M or less. Even more preferably, the isolated human
antibody, or an antigen-binding portion thereof, dissociates from human
IL-12 with a Koff rate constant of 1×10-5 s-1 or
less, or inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-11 M or less.

[0019] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics:

[0020] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-6 M or less;

[0038] In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR2 comprising the
amino acid sequence selected from the group consisting of SEQ ID
NO:335-SEQ ID NO: 403; and a light chain CDR2 comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 506-SEQ ID NO:
533. In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR1 comprising the
amino acid sequence selected from the group consisting of SEQ ID NO:
288-SEQ ID NO: 334; and a light chain CDR1 comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 470-SEQ ID NO:
505. In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, comprising a the heavy chain variable
region comprising the amino acid sequence of SEQ ID NO: 23, and a light
chain variable region comprising the amino acid sequence of SEQ ID NO:
24. In a preferred embodiment, the isolated human antibody comprises a
heavy chain constant region, or an Fab fragment or a F(ab')2
fragment or a single chain Fv fragment as described above.

[0039] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which

[0040] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0043] In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, has a heavy chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 27; and a light chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 28. In a preferred embodiment, the
isolated human antibody, or an antigen-binding portion thereof, has a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29; and
a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 30.
In a preferred embodiment, the isolated human antibody, or an
antigen-binding portion thereof, which has a heavy chain variable region
comprising the amino acid sequence of SEQ ID NO: 31, and a light chain
variable region comprising the amino acid sequence of SEQ ID NO: 32. In a
preferred embodiment, the isolated human antibody comprises a heavy chain
constant region, or an Fab fragment, or a F(ab')2 fragment or a
single chain Fv fragment as described above.

[0044] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which

[0045] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-6 M or less;

[0046] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO:5, or a mutant thereof having one or more amino acid
substitutions at a contact position or a hypermutation position, wherein
said mutant has a koff rate no more than 10-fold higher than the
antibody comprising a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 3, and a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 5; and

[0047] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 6, or a mutant thereof having one or more amino acid
substitutions at a contact position or a hypermutation position, wherein
said mutant has a koff rate no more than 10-fold higher than the
antibody comprising a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 6.

[0048] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which

[0049] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0050] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 13, or a mutant thereof having one or more amino acid
substitutions at a contact position or a hypermutation position, wherein
said mutant has a koff rate no more than 10-fold higher than the
antibody comprising a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 11, and a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 13; and

[0051] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 14, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position, contact
position or a hypermutation position, wherein said mutant has a koff
rate no more than 10-fold higher than the antibody comprising a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0052] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which

[0053] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0054] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 21, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position, contact
position or a hypermutation position, wherein said mutant has a koff
rate no more than 10-fold higher than the antibody comprising a heavy
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17, a heavy
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 19, and a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21; and

[0062] In another aspect, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0063] a) that binds to human IL-12 and dissociates
from human IL-12 with a koff rate constant of 0.1 s-1 or less,
as determined by surface plasmon resonance, or which inhibits
phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin
blast proliferation assay (PHA assay) with an IC50 of
1×10-6M or less. [0064] b) has a heavy chain variable region
comprising an amino acid sequence selected from a member of the VH3
germline family, wherein the heavy chain variable region has a mutation
at a preferred selective mutagenesis position, contact or hypermutation
position with an activity enhancing amino acid residue. [0065] c) has a
light chain variable region comprising an amino acid sequence selected
from a member of the V.sub.λ1 germline family, wherein the light
chain variable region has a mutation at a preferred selective mutagenesis
position, contact position or hypermutation position with an activity
enhancing amino acid residue.

[0066] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0067] a) that binds to human IL-12 and dissociates
from human IL-12 with a koff rate constant of 0.1 s-1 or less,
as determined by surface plasmon resonance, or which inhibits
phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin
blast proliferation assay (PHA assay) with an IC50 of
1×10-6M or less. [0068] b) has a heavy chain variable region
comprising an amino acid sequence selected from the group consisting of
SEQ ID NOs: 595-667, wherein the heavy chain variable region has a
mutation at a preferred selective mutagenesis position, contact position
or hypermutation position with an activity enhancing amino acid residue.
[0069] c) has a light chain variable region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 669-675,
wherein the light chain variable region has a mutation at a preferred
selective mutagenesis position, contact or hypermutation position with an
activity enhancing amino acid residue.

[0070] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0071] a) that binds to human IL-12 and dissociates
from human IL-12 with a koff rate constant of 0.1 s-1 or less,
as determined by surface plasmon resonance, or which inhibits
phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin
blast proliferation assay (PHA assay) with an IC50 of
1×10-6M or less. [0072] b) has a heavy chain variable region
comprising the COS-3 germline amino acid sequence, wherein the heavy
chain variable region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity enhancing
amino acid residue. [0073] c) has a light chain variable region
comprising the DPL8 germline amino acid sequence, wherein the light chain
variable region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity enhancing
amino acid residue.

[0074] In another embodiment, the invention provides an isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0075] a) that binds to human IL-12 and dissociates
from human IL-12 with a koff rate constant of 0.1 s-1 or less,
as determined by surface plasmon resonance, or which inhibits
phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin
blast proliferation assay (PHA assay) with an IC50 of
1×10-6M or less. [0076] b) has a heavy chain variable region
comprising an amino acid sequence selected from a member of the VH3
germline family, wherein the heavy chain variable region comprises a CDR2
that is structurally similar to CDR2s from other VH3 germline family
members, and a CDR1 that is structurally similar to CDR1s from other
VH3 germline family members, and wherein the heavy chain variable
region has a mutation at a preferred selective mutagenesis position,
contact or hypermutation position with an activity enhancing amino acid
residue; [0077] c) has a light chain variable region comprising an amino
acid sequence selected from a member of the V.sub.λ1 germline
family, wherein the light chain variable region comprises a CDR2 that is
structurally similar to CDR2s from other V.sub.λ1 germline family
members, and a CDR1 that is structurally similar to CDR1s from other
V.sub.λ1 germline family members, and wherein the light chain
variable region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity enhancing
amino acid residue.

[0078] In a preferred embodiment, the isolated human antibody, or antigen
binding portion thereof, has a mutation in the heavy chain CDR3. In
another preferred embodiment, the isolated human antibody, or antigen
binding portion thereof, has a mutation in the light chain CDR3. In
another embodiment, the isolated human antibody, or antigen binding
portion thereof, has a mutation in the heavy chain CDR2. In another
preferred embodiment, the isolated human antibody, or antigen binding
portion thereof, has a mutation in the light chain CDR2. In another
preferred embodiment, the isolated human antibody, or antigen binding
portion thereof, has a mutation in the heavy chain CDR1. In another
preferred embodiment, the isolated human antibody, or antigen binding
portion thereof, has a mutation in the light chain CDR1.

[0079] In another aspect, the invention provides recombinant expression
vectors carrying the antibody-encoding nucleic acids of the invention,
and host cells into which such vectors have been introduced, are also
encompassed by the invention, as are methods of making the antibodies of
the invention by culturing the host cells of the invention.

[0080] In another aspect, the invention provides an isolated human
antibody, or antigen-binding portion thereof, that neutralizes the
activity of human IL-12, and at least one additional primate IL-12
selected from the group consisting of baboon IL-12, marmoset IL-12,
chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which does not
neutralize the activity of the mouse IL-12.

[0081] In another aspect, the invention provides a pharmaceutical
composition comprising the antibody or an antigen binding portion
thereof, of the invention and a pharmaceutically acceptable carrier.

[0082] In another aspect, the invention provides a composition comprising
the antibody or an antigen binding portion thereof, and an additional
agent, for example, a therapeutic agent.

[0083] In another aspect, the invention provides a method for inhibiting
human IL-12 activity comprising contacting human IL-12 with the antibody
of the invention, e.g., J695, such that human IL-12 activity is
inhibited.

[0084] In another aspect, the invention provides a method for inhibiting
human IL-12 activity in a human subject suffering from a disorder in
which IL-12 activity is detrimental, comprising administering to the
human subject the antibody of the invention, e.g., J695, such that human
IL-12 activity in the human subject is inhibited. The disorder can be,
for example, Crohn's disease, multiple sclerosis or rheumatoid arthritis.

[0085] In another aspect, the invention features a method for improving
the activity of an antibody, or an antigen binding portion thereof, to
attain a predetermined target activity, comprising:

[0088] c) individually mutating the selected preferred selective
mutagenesis position to at least two other amino acid residues to hereby
create a first panel of mutated antibodies, or antigen binding portions
thereof;

[0089] d) evaluating the activity of the first panel of mutated
antibodies, or antigen binding portions thereof to determined if mutation
of a single selective mutagenesis position produces an antibody or
antigen binding portion thereof with the predetermined target activity or
a partial target activity;

[0090] e) combining in a stepwise fashion, in the parent antibody, or
antigen binding portion thereof, individual mutations shown to have an
improved activity, to form combination antibodies, or antigen binding
portions thereof.

[0091] f) evaluating the activity of the combination antibodies, or
antigen binding portions thereof to determined if the combination
antibodies, or antigen binding portions thereof have the predetermined
target activity or a partial target activity.

[0092] g) if steps d) or f) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity, or
result an antibody with only a partial activity, additional amino acid
residues selected from the group consisting of H35, H50, H53, H54, H95,
H96, H97, H98, L30A and L96 are mutated to at least two other amino acid
residues to thereby create a second panel of mutated antibodies or
antigen-binding portions thereof;

[0093] h) evaluating the activity of the second panel of mutated
antibodies or antigen binding portions thereof, to determined if mutation
of a single amino acid residue selected from the group consisting of H35,
H50, H53, H54, H95, H96, H97, H98, L30A and L96 results an antibody or
antigen binding portion thereof, having the predetermined target activity
or a partial activity;

[0094] i) combining in stepwise fashion in the parent antibody, or
antigen-binding portion thereof, individual mutations of step g) shown to
have an improved activity, to form combination antibodies, or antigen
binding portions thereof;

[0095] j) evaluating the activity of the combination antibodies or antigen
binding portions thereof, to determined if the combination antibodies, or
antigen binding portions thereof have the predetermined target activity
or a partial target activity;

[0096] k) if steps h) or j) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity, or
result in an antibody with only a partial activity, additional amino acid
residues selected from the group consisting of H33B, H52B and L31A are
mutated to at least two other amino acid residues to thereby create a
third panel of mutated antibodies or antigen binding portions thereof;

[0097] l) evaluating the activity of the third panel of mutated antibodies
or antigen binding portions thereof, to determine if a mutation of a
single amino acid residue selected from the group consisting of H33B,
H52B and L31A resulted in an antibody or antigen binding portion thereof,
having the predetermined target activity or a partial activity;

[0098] m) combining in a stepwise fashion in the parent antibody, or
antigen binding portion thereof, individual mutation of step k) shown to
have an improved activity, to form combination antibodies, or antigen
binding portions, thereof;

[0099] n) evaluating the activity of the combination antibodies or
antigen-binding portions thereof, to determine if the combination
antibodies, or antigen binding portions thereof have the predetermined
target activity to thereby produce an antibody or antigen binding portion
thereof with a predetermined target activity.

[0100] In another aspect, the invention provides a method for improving
the activity of an antibody, or antigen-binding portion thereof,
comprising:

[0103] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;

[0104] d) evaluating the activity of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof;

[0105] e) repeating steps b) through d) for at least one other contact or
hypermutation position;

[0106] f) combining, in the parent antibody, or antigen-binding portion
thereof, individual mutations shown to have improved activity, to form
combination antibodies, or antigen-binding portions thereof; and

[0107] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0108] In one embodiment, the invention provides a method for improving
the activity of an antibody, or antigen-binding portion thereof,
comprising:

[0109] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity is not further improved by mutagenesis in said
phage-display system;

[0111] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0112] d) evaluating the activity of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof;

[0113] e) repeating steps b) through d) for at least one other contact or
hypermutation position;

[0114] f) combining, in the parent antibody, or antigen-binding portion
thereof, individual mutations shown to have improved activity, to form
combination antibodies, or antigen-binding portions thereof; and

[0115] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0116] In a preferred embodiment, the contact positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96. In another preferred
embodiment, the hypermutation positions are selected from the group
consisting of H30, H31, H31B, H32, H52, H56, H58, L30, L31, L32, L53 and
L93. In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94. In a more preferred
embodiment, the contact positions are selected from the group consisting
of L50 and L94.

[0117] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0120] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expressing said panel
in an appropriate expression system;

[0122] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristics,
wherein the property or characteristic is one that needs to be retained
in the antibody;

until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0123] In a preferred embodiment, the contact positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence. In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52, H56,
H58, L30, L31, L32, L53 and L93 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment the residues for selective mutagenesis are
selected from the preferred selective mutagenesis positions from the
group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment, the contact positions are selected from the
group consisting of L50 and L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0124] In another embodiment of the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0125] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0127] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0129] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristic,
wherein the property or characteristic is one that needs to be retained,
until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0130] f) repeating steps a) through e) for at least one other preferred
selective mutagenesis position, contact or hypermutation position;

[0131] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and at least on retained property or
characteristic, to form combination antibodies, or antigen-binding
portions thereof; and

[0132] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof, until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one retained
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0133] In a preferred embodiment, the contact positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence. In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52, H56,
H58, L30, L31, L32, L53 and L93 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment the residues for selective mutagenesis are
selected from the preferred selective mutagenesis positions from the
group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment, the contact positions are selected from the
group consisting of L50 and L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0134] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0135] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0136] b) selecting a contact or hypermutation position within a
complementarity determining region (CDR) for mutation, thereby
identifying a selected contact or hypermutation position;

[0137] c) individually mutating said selected contact or hypermutation
position to at least two other amino acid residues to thereby create a
panel of mutated antibodies, or antigen-binding portions thereof, and
expressing said panel in a non-phage display system;

[0139] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristics,
wherein the property or characteristic is one that needs to be retained;

until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0140] In a preferred embodiment, the contact positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence. In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52, H56,
H58, L30, L31, L32, L53 and L93 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment the residues for selective mutagenesis are
selected from the preferred selective mutagenesis positions from the
group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment, the contact positions are selected from the
group consisting of L50 and L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0141] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0142] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0144] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0146] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristic,
wherein the property or characteristic is one that needs to be retained,
until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0147] f) repeating steps a) through e) for at least one other preferred
selective mutagenesis position, contact or hypermutation position;

[0148] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and at least on retained other
characteristic, to form combination antibodies, or antigen-binding
portions thereof; and

[0149] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one retained
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0150] In a preferred embodiment, the contact positions are selected from
the group consisting of H30, H31, H31B, H32, H33, H35, H50, H52, H52A,
H53, H54, H56, H58, H95, H96, H97, H98, H101, L30, L31, L32, L34, L50,
L52, L53, L55, L91, L92, L93, L94 and L96 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence. In another preferred embodiment, the hypermutation positions
are selected from the group consisting of H30, H31, H31B, H32, H52, H56,
H58, L30, L31, L32, L53 and L93 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment the residues for selective mutagenesis are
selected from the preferred selective mutagenesis positions from the
group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence. In
a more preferred embodiment, the contact positions are selected from the
group consisting of L50 and L94 and the other characteristic is selected
from 1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0151] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0156] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic;

until an antibody, or antigen-binding portion thereof, with an improved
activity, relative to the parent antibody, or antigen-binding portion
thereof, is obtained.

[0157] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0158] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0164] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity, to form combination antibodies, or
antigen-binding portions thereof; and

[0165] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.

[0166] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0167] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0169] c) individually mutating said selected contact or hypermutation
position to at least two other amino acid residues to thereby create a
panel of mutated antibodies, or antigen-binding portions thereof, and
expressing said panel in a non-phage display system;

[0171] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic until an antibody, or antigen-binding portion thereof,
with an improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0172] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0173] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0174] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0176] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0179] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity, to form combination antibodies, or
antigen-binding portions thereof; and

[0180] g) evaluating the activity and other property or characteristic of
the combination antibodies, or antigen-binding portions thereof with two
activity enhancing amino acid residues, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0181] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0182] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0187] e) evaluating the panel of mutated antibodies or antigen-binding
portions thereof, relative to the parent antibody or antigen-portion
thereof, for changes in at least one other property or characteristic;

[0189] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and not affecting at least one other
property or characteristic, to form combination antibodies, or
antigen-binding portions thereof; and

[0190] h) evaluating the activity and the retention of at least one other
characteristic or property of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one retained property or characteristic,
relative to the parent antibody, or antigen-binding portion thereof, is
obtained.

[0191] In another embodiment the invention provides a method to improve
the affinity of an antibody or antigen-binding portion thereof,
comprising:

[0192] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0194] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0196] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other characteristic or
property until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.

[0197] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0202] e) evaluating the panel of mutated antibodies or antigen-binding
portions thereof, relative to the parent antibody or antigen-portion
thereof, for changes in at least one other property or characteristic;

[0204] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity but not affecting at least one other
property or characteristic, to form combination antibodies, or
antigen-binding portions thereof with at least one retained property or
characteristic; and

[0205] h) evaluating the activity and the retention of at least one
property of characteristic of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one retained property or characteristic,
relative to the parent antibody, or antigen-binding portion thereof, is
obtained.

[0206] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0207] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, without affecting other characteristics, comprising:

[0212] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic until an antibody, or antigen-binding portion thereof,
with an improved activity and retained other characteristic or property,
relative to the parent antibody, or antigen-binding portion thereof, is
obtained.

[0213] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0214] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0216] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0217] d) evaluating the activity and retention of at least one other
characteristic or property of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof, thereby identifying an activity
enhancing amino acid residue;

[0219] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and not to affect at least one other
characteristic or property, to form combination antibodies, or
antigen-binding portions thereof; and

[0220] g) evaluating the activity and retention of at least one other
characteristic or property of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one other retained characteristic or
property, relative to the parent antibody, or antigen-binding portion
thereof, is obtained.

[0221] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0222] In yet another aspect, the invention provides an isolated antibody
(e.g., a human antibody) or antigen binding portion thereof that is
capable of binding to the p40 subunit of IL-12 (e.g., the p40 subunit of
human IL-12) and is capable of altering the conformational structure of
said p40 subunit of IL-12.

[0223] In a related aspect, the invention provides an isolated antibody
(e.g., a human antibody) or antigen binding portion thereof that is
capable of binding to the p40 subunit of IL-12 (e.g., the p40 subunit of
human IL-12) and is capable of altering the conformational structure of
an interleukin, e.g., the p40 subunit of an interleukin. In one
embodiment, the interleukin is IL-12. In another embodiment, the
interleukin comprises a p40 subunit and a p19 subunit, e.g., the
interleukin is IL-23. In yet another embodiment, the interleukin is a
human interleukin such as human IL-12 or human IL-23.

[0224] In one embodiment, the antibody is Y61 or J695. In another
embodiment, the antibody is not Y61 or J695.

[0225] In another embodiment, the isolated antibody, or antigen binding
portion thereof, alters the conformational structure of the interleukin,
e.g., IL-12 or IL-23, such that binding by the interleukin, e.g., IL-12
or IL-23, to an interleukin-interacting molecule is modulated.

[0226] In yet another embodiment, the interleukin-interacting molecule is
an interleukin receptor, e.g., IL-12 or IL-23 receptor.

[0227] In a further embodiment, the isolated antibody, or antigen binding
portion thereof, alters the conformational structure of an interleukin,
e.g., IL-12 or IL-23, such that binding to the interleukin, e.g., IL-12
or IL-23, by a second antibody is inhibited.

[0228] In one embodiment, the interleukin is IL-12 and the second antibody
binds to an epitope of the p40 subunit of IL-12 to which an antibody
selected from the group consisting of 1D4.7, C8.6.2 and C340 binds.

[0229] In another embodiment, the interleukin is IL-12 and the second
antibody is selected from the group consisting of 1D4.7, C8.6.2 and C340.

[0230] In another aspect, the invention provides an isolated antibody,
e.g., a human antibody, or antigen binding portion thereof, that binds to
an epitope of the p40 subunit of IL-12 to which an antibody selected from
the group consisting of 1D4.7, C8.6.2, C340 and 7G3 does not bind.

[0231] In one embodiment, the antibody is Y61 or J695. In another
embodiment, the antibody is not Y61 or J695.

[0232] In another embodiment, the isolated antibody, or antigen binding
portion thereof, binds to an epitope of the p40 subunit of IL-12 to which
an antibody selected from the group consisting of Y61 and J695 binds.

[0233] In one embodiment, the antibody is not Y61 or J695.

[0234] In a further embodiment, the isolated antibody, or antigen binding
portion thereof, inhibits the binding to IL-12 by a second antibody.

[0235] In yet a further embodiment, the second antibody binds to an
epitope of IL-12 to which an antibody selected from the group consisting
of 1D4.7, C8.6.2, C340 binds.

[0236] In one embodiment, the second antibody is selected from the group
consisting of 1D4.7, C8.6.2 and C340.

[0237] In one embodiment, the isolated antibody, or antigen binding
portion thereof, dissociates from the p40 subunit of human IL-12 with a
Kd of 1×10-10 M or less or a koff rate constant of
1×10-3 s-1 or less, as determined by surface plasmon
resonance.

[0238] In another embodiment, the isolated antibody, or antigen binding
portion thereof, is a neutralizing antibody

[0239] In a further embodiment, the isolated antibody, or antigen binding
portion thereof, inhibits phytohemagglutinin blast proliferation in an in
vitro PHA assay with an IC50 of 1×10-9 M or less, or
which inhibits human IFNγ production with an IC50 of
1×10-10 M or less.

[0240] In one embodiment, the isolated antibody, or antigen binding
portion thereof, is a human antibody.

[0244] In another aspect, the invention provides a composition comprising
an antibody (e.g., a human antibody) or antigen binding portion thereof,
that is capable of binding to the p40 subunit of IL-12 and is capable of
altering the conformational structure of an interleukin comprising a p40
subunit, e.g., IL-12 or IL-23, in an amount effective for altering the
conformational structure of the interleukin. In one embodiment, the
interleukin is IL-12. In another embodiment, the interleukin comprises a
p40 subunit and a p19 subunit, e.g., the interleukin is IL-23.

[0245] In one embodiment, the amount of the antibody effective to alter
the conformational structure of the p40 subunit of an interleukin, e.g.,
IL-12, is between about 0.1 and 2500 μg/ml, preferably between about
1.0 and 250 μg/ml, more preferably between about 2.0 and 125 μg/ml,
more preferably between about 4.0 and 65 μg/ml, even more preferably
between about 6.0 and 50 μg/ml, preferably between about 8.0 and 40
μg/ml, and most preferably between about 10 and 25 μg/ml. In
various embodiments, the amount of the antibody effective to alter the
conformational structure of the p40 subunit of an interleukin, e.g.,
IL-12, is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,
35, 36, 37, 38, 39, 40, 45, 50, 55, 60 μg/ml or more. Ranges
intermediate to the above recited amounts, e.g., between about 5.0 and 55
μg/ml, between about 9.0 and 35 μg/ml, and between about 15 and 20
μg/ml, are also intended to be part of this invention. For example,
ranges of values using a combination of any of the above recited values
as upper and/or lower limits are intended to be included. Further,
discrete amounts intermediate to any of the above recited amounts, e.g.,
10.5, 11.5, and 12.5 μg/ml, are also intended to be part of this
invention.

[0246] In another aspect, the invention provides a composition comprising
an antibody (e.g., a human antibody) or antigen binding portion thereof,
that binds to the p40 subunit of IL-12 and is capable of altering the
conformational structure of said p40 subunit packaged or promoted with
instructions for use in altering the conformational structure of said p40
subunit.

[0247] In a related aspect, the invention provides a composition
comprising an antibody (e.g., a human antibody) or antigen binding
portion thereof, that binds to the p40 subunit of IL-12 and is capable of
altering the conformational structure of an interleukin comprising a p40
subunit, e.g., IL-12 or IL-23, packaged or promoted with instructions for
use in altering the conformational structure of the interleukin, e.g.,
IL-12 or IL-23.

[0248] In one embodiment, the composition further comprises a means for
determining whether the conformational structure of the interleukin,
e.g., IL-12 or IL-23, has been altered.

[0249] In one embodiment, the interleukin is IL-12 and the antibody, or
antigen binding portion thereof, binds to an epitope of the p40 subunit
of IL-12 to which an antibody selected from the group consisting of
1D4.7, C8.6.2, C340 and 7G3 does not bind.

[0250] In another embodiment, the antibody, or antigen binding portion
thereof, binds an epitope to which an antibody selected from the group
consisting of Y61 and J695 binds.

[0251] In yet another embodiment, the antibody, or antigen binding portion
thereof, is not Y61 or J695.

[0252] In a further embodiment, the antibody, or antigen binding portion
thereof, dissociates from the p40 subunit of human IL-12 with a Kd
of 1×10-1M or less or a koff rate constant of
1×10-3 s-1 or less, as determined by surface plasmon
resonance.

[0253] In one embodiment, the antibody, or antigen binding portion
thereof, is a neutralizing antibody.

[0254] In one embodiment, the antibody, or antigen binding portion
thereof, is a human antibody.

[0255] In yet another aspect, the invention provides a composition
comprising a plurality of antibodies, or antigen binding portions
thereof, wherein each antibody of the plurality of antibodies binds to a
different epitope of the p40 subunit of IL-12.

[0256] In one embodiment, at least one antibody, or antigen binding
portion thereof, binds to an epitope of the p40 subunit of IL-12 to which
an antibody selected from the group consisting of Y61 and J695 binds.

[0257] In another embodiment, at least one antibody, or antigen binding
portion thereof, binds to an epitope of the p40 subunit of IL-12 to which
an antibody selected from the group consisting of 1D4.7, C8.6.2, C340 and
7G3 does not bind.

[0258] In one embodiment, at least one antibody, or antigen binding
portion thereof, dissociates from the p40 subunit of human IL-12 with a
Kd of 1×10-10 M or less or a koff rate constant of
1×10-3 s-1 or less, as determined by surface plasmon
resonance.

[0259] In another embodiment, at least one antibody, or antigen binding
portion thereof, is a neutralizing antibody.

[0260] In yet another embodiment, at least one antibody, or antigen
binding portion thereof, inhibits phytohemagglutinin blast proliferation
in an in vitro PHA assay with an IC50 of 1×10-9 M or
less, or which inhibits human IFNγ production with an IC50 of
1×10-10 M or less.

[0261] In a further embodiment, at least one antibody, or antigen binding
portion thereof, is a human antibody.

[0262] In one embodiment, at least one antibody, or antigen binding
portion thereof, has a heavy chain CDR3 comprising the amino acid
sequence of SEQ ID NO: 25 and a light chain CDR3 comprising the amino
acid sequence of SEQ ID NO: 26.

[0263] In another embodiment, the at least one antibody, or antigen
binding portion thereof, further has a heavy chain CDR2 comprising the
amino acid sequence of SEQ ID NO: 27 and a light chain CDR2 comprising
the amino acid sequence of SEQ ID NO: 28.

[0264] In yet another embodiment, the at least one antibody, or antigen
binding portion thereof, further has a heavy chain CDR1 comprising the
amino acid sequence of SEQ ID NO: 29 and a light chain CDR1 comprising
the amino acid sequence of SEQ ID NO: 30.

[0265] In one aspect, the invention provides a method for detecting the
p40 subunit of an interleukin, e.g., IL-12 or IL-23, comprising
contacting the p40 subunit of IL-12 with an antibody of the invention, or
antigen-binding portion thereof, such that the p40 subunit is detected.

[0266] In a related aspect, the invention provides a method for detecting
the p40 subunit of an interleukin, e.g., IL-12 or IL-23, comprising
contacting the p40 subunit of the interleukin, e.g., IL-12 or IL-23, with
an antibody of the invention, or antigen-binding portion thereof, such
that the p40 subunit of the interleukin, e.g., IL-12 or IL-23, is
detected.

[0267] In one embodiment, the p40 subunit of the interleukin, e.g., IL-12
or IL-23, is detected in vitro. In another embodiment, the p40 subunit of
the interleukin, e.g., IL-12 or IL-23, is detected in a biological sample
for diagnostic purposes.

[0268] In another aspect, the invention provides a method for inhibiting
an activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, comprising contacting the interleukin, e.g., IL-12 or IL-23, with
an antibody, or antigen-binding portion thereof, of the invention such
that the activity is inhibited.

[0269] In a related aspect, the invention provides a method for inhibiting
an activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, in a human subject suffering from a disorder in which the activity
is detrimental, comprising administering to the human subject an
antibody, or antigen-binding portion thereof, of the invention such that
the activity of the interleukin, e.g., IL-12 or IL-23, in the human
subject is inhibited.

[0270] In one embodiment, the interleukin is IL-12. In another embodiment,
the interleukin comprises a p40 subunit and a p19 subunit, e.g., the
interleukin is IL-23.

[0276] In yet another aspect, the invention provides a method for altering
the conformational structure of the p40 subunit of IL-12, the method
comprising contacting said subunit with an antibody, or antigen binding
portion thereof, that is capable of binding said subunit and is capable
of altering the conformational structure of said subunit, in an amount
effective to alter the conformational structure of said subunit, thereby
altering the conformational structure of said subunit.

[0277] In a related aspect, the invention provides a method for altering
the conformational structure of an interleukin comprising a p40 subunit,
e.g., IL-12 or IL-23, the method comprising contacting the interleukin,
e.g., IL-12 or IL-23, with an antibody, or antigen binding portion
thereof, that is capable of binding to the p40 subunit of IL-12 and is
capable of altering the conformational structure of the interleukin,
e.g., IL-12 or IL-23, in an amount effective to alter the conformational
structure of the interleukin, e.g., IL-12 or IL-23, thereby altering the
conformational structure of the interleukin, e.g., IL-12 or IL-23.

[0278] In another aspect, the invention provides a method for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, the method comprising contacting the interleukin, e.g., IL-12 or
IL-23, with an antibody, or antigen binding portion thereof, that is
capable of binding to the p40 subunit of IL-12 and is capable of altering
the conformational structure of the said subunit, in an amount effective
to alter the conformational structure of said subunit, thereby inhibiting
the activity of the interleukin, e.g., IL-12 or IL-23.

[0279] In a related aspect, the invention provides a method for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, the method comprising contacting the interleukin, e.g., IL-12 or
IL-23, with an antibody, or antigen binding portion thereof, that is
capable of binding to the p40 subunit of IL-12 and is capable of altering
the conformational structure of the interleukin, e.g., IL-12 or IL-23, in
an amount effective to alter the conformational structure of the
interleukin, e.g., IL-12 or IL-23, thereby inhibiting the activity of the
interleukin, e.g., IL-12 or IL-23.

[0280] In another aspect, the invention provides a method for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, in a human subject suffering from a disorder in which the activity
of the interleukin, e.g., IL-12 or IL-23, is detrimental, the method
comprising administering to the human subject an antibody, or antigen
binding portion thereof, that is capable of binding to the p40 subunit of
IL-12 and is capable of altering the conformational structure of said
subunit, in an amount effective to alter the conformational structure of
said subunit, thereby inhibiting the activity of the interleukin, e.g.,
IL-12 or IL-23, in the human subject.

[0281] In a related aspect, the invention provides a method for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, in a human subject suffering from a disorder in which the activity
of the interleukin, e.g., IL-12 or IL-23, is detrimental, the method
comprising administering to the human subject an antibody, or antigen
binding portion thereof, that is capable of binding to the p40 subunit of
IL-12 and is capable of altering the conformational structure of the
interleukin, e.g., IL-12 or IL-23, in an amount effective to alter the
conformational structure of the interleukin, e.g., IL-12 or IL-23,
thereby inhibiting the activity of the interleukin, e.g., IL-12 or IL-23,
in the human subject.

[0282] In one embodiment, the interleukin is IL-12. In another embodiment,
the interleukin comprises a p40 subunit and a p19 subunit, e.g., the
interleukin is IL-23.

[0289] In another embodiment, the amount of the antibody effective to
alter the conformational structure of the p40 subunit of an interleukin,
e.g., IL-12, is between about 0.1 and 2500 μg/ml, preferably between
about 1.0 and 250 μg/ml, more preferably between about 2.0 and 125
μg/ml, more preferably between about 4.0 and 65 μg/ml, even more
preferably between about 6.0 and 50 μg/ml, preferably between about
8.0 and 40 μg/ml, and most preferably between about 10 and 25
μg/ml. In various embodiments, the amount of the antibody effective to
alter the conformational structure of the p40 subunit of an interleukin,
e.g., IL-12, is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,
34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60 μg/ml or more. Ranges
intermediate to the above recited amounts, e.g., between about 5.0 and 55
μg/ml, between about 9.0 and 35 μg/ml, and between about 15 and 20
μg/ml, are also intended to be part of this invention. For example,
ranges of values using a combination of any of the above recited values
as upper and/or lower limits are intended to be included. Further,
discrete amounts intermediate to any of the above recited amounts, e.g.,
10.5, 11.5, and 12.5 μg/ml, are also intended to be part of this
invention.

[0291] In another aspect, the invention provides a method for identifying
an antibody, or antigen binding portion thereof, suitable for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23, the method comprising contacting the p40 subunit of an
interleukin, e.g., IL-12 or IL-23, or a portion thereof, with an
antibody, or antigen binding portion thereof, and determining whether the
antibody, or antigen binding portion thereof, alters the conformational
structure of said p40 subunit, thereby identifying an antibody, or
antigen binding portion thereof, suitable for inhibiting the activity of
an interleukin comprising a p40 subunit, e.g., IL-12 or IL-23.

[0292] In a related aspect, the invention provides a method for
identifying an antibody, or antigen binding portion thereof, suitable for
inhibiting the activity of an interleukin comprising a p40 subunit, e.g.,
IL-12 or IL-23, the method comprising contacting the p40 subunit of an
interleukin, e.g., IL-12 or IL-23, or a portion thereof, with an
antibody, or antigen binding portion thereof, and determining whether the
antibody, or antigen binding portion thereof, alters the conformational
structure of the interleukin, e.g., IL-12 or IL-23, thereby identifying
an antibody, or antigen binding portion thereof, suitable for inhibiting
the activity of an interleukin comprising a p40 subunit, e.g., IL-12 or
IL-23.

[0293] In one embodiment, the antibody, or antigen binding portion
thereof, is suitable for inhibiting the activity of the interleukin,
e.g., IL-12 or IL-23, in a human subject suffering from a disorder in
which the activity of the interleukin, e.g., IL-12 or IL-23; is
detrimental.

[0294] In another embodiment, the method further comprises determining
whether binding by the interleukin, e.g., IL-12 or IL-23, to an
interleukin-interacting molecule is modulated.

[0295] In one embodiment, the molecule that interacts with the
interleukin, e.g., IL-12 or IL-23, is an interleukin receptor, e.g.,
IL-12 receptor or IL-23 receptor.

[0296] In one embodiment, the method further comprises determining whether
binding to the interleukin, e.g., IL-12 or IL-23, by a second antibody is
inhibited.

[0297] In one embodiment, the interleukin is IL-12 and the second antibody
binds to an epitope of the p40 subunit of IL-12 to which an antibody
selected from the group consisting of 1D4.7, C8.6.2 and C340 binds.

[0298] In one embodiment, the second antibody is selected from the group
consisting of 1D4.7, C8.6.2 and C340.

BRIEF DESCRIPTION OF THE DRAWINGS

[0299] FIGS. 1A-1B show the heavy chain variable region amino acid
sequence alignments of a series of human antibodies that bind human IL-12
compared to germline sequences Cos-3/JH3 and Dpl18 Lv1042. Kabat
numbering is used to identify amino acid positions. For the Joe 9 wild
type, the full sequence is shown. For the other antibodies, only those
amino acids positions that differ from Joe 9 wild type are shown.

[0300] FIGS. 1C-1D show the light chain variable region amino acid
sequence alignments of a series of human antibodies that bind human
IL-12. Kabat numbering is used to identify amino acid positions. For the
Joe 9 wild type, the full sequence is shown. For the other antibodies,
only those amino acids positions that differ from Joe 9 wild type are
shown.

[0301] FIGS. 2A-2E show the CDR positions in the heavy chain of the Y61
antibody that were mutated by site-directed mutagenesis and the
respective amino acid substitutions at each position. The graphs at the
right of the figures show the off-rates for the substituted antibodies
(black bars) as compared to unmutated Y61 (open bar).

[0302] FIGS. 2F-2H show the CDR positions in the light chain of the Y61
antibody; that were mutated by site-directed mutagenesis and the
respective amino acid substitutions at each position. The graphs at the
right of the figures show the off-rates for the substituted antibodies
(black bars) as compared to unmutated Y61 (open bar).

[0304]FIG. 4 shows a graph of mean arthritic score versus days after
immunization of mice with collagen, demonstrating that treatment with
C17.15 significantly decreases arthritis-related symptoms as compared to
treatment with rat IgG.

DETAILED DESCRIPTION OF THE INVENTION

[0305] In order that the present invention may be more readily understood,
certain terms are first defined.

[0306] The term "activity enhancing amino acid residue" includes an amino
acid residue which improves the activity of the antibody. It should be
understood that the activity enhancing amino acid residue may replace an
amino acid residue at a contact, hypermutation or preferred selective
mutagenesis position and, further, more than one activity enhancing amino
acid residue can be present within one or more CDRs. An activity
enhancing amino acid residue include, an amino acid residue that improves
the binding specificity/affinity of an antibody, for example anti-human
IL-12 antibody binding to human IL-12. The activity enhancing amino acid
residue is also intended to include an amino acid residue that improves
the neutralization potency of an antibody, for example, the human IL-12
antibody which inhibits human IL-12.

[0307] The term "antibody" includes an immunoglobulin molecule comprised
of four polypeptide chains, two heavy (H) chains and two light (L) chains
inter-connected by disulfide bonds. Each heavy chain is comprised of a
heavy chain variable region (abbreviated herein as HCVR or VH) and a
heavy chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised of a
light chain variable region (abbreviated herein as LCVR or VL) and a
light chain constant region. The light chain constant region is comprised
of one domain, CL. The VH and VL regions can be further subdivided into
regions of hypervariability, termed complementarity determining regions
(CDRs), interspersed with regions that are more conserved, termed
framework regions (FR). Each VH and VL is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0308] The term "antigen-binding portion" of an antibody (or "antibody
portion") includes fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., hIL-12). It has been shown that
the antigen-binding function of an antibody can be performed by fragments
of a full-length antibody. Examples of binding fragments encompassed
within the term "antigen-binding portion" of an antibody include (i) a
Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1
domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two
Fab fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment
consisting of the VL and VH domains of a single arm of an antibody, (v) a
dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of
a VH domain; and (vi) an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment, VL and
VH, are coded for by separate genes, they can be joined, using
recombinant methods, by a synthetic linker that enables them to be made
as a single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et
al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding portion" of
an antibody. Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies in
which VH and VL domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the two
domains on the same chain, thereby forcing the domains to pair with
complementary domains of another chain and creating two antigen binding
sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123). Still
further, an antibody or antigen-binding portion thereof may be part of a
larger immunoadhesion molecules, formed by covalent or non-covalent
association of the antibody or antibody portion with one or more other
proteins or peptides. Examples of such immunoadhesion molecules include
use of the streptavidin core region to make a tetrameric scFv molecule
(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas
6:93-101) and use of a cysteine residue, a marker peptide and a
C-terminal polyhistidine tag to make bivalent and biotinylated scFv
molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).
Antibody portions, such as Fab and F(ab')2 fragments, can be
prepared from whole antibodies using conventional techniques, such as
papain or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described herein.
Preferred antigen binding portions are complete domains or pairs of
complete domains.

[0309] The term "backmutation" refers to a process in which some or all of
the somatically mutated amino acids of a human antibody are replaced with
the corresponding germline residues from a homologous germline antibody
sequence. The heavy and light chain sequences of the human antibody of
the invention are aligned separately with the germline sequences in the
VBASE database to identify the sequences with the highest homology.
Differences in the human antibody of the invention are returned to the
germline sequence by mutating defined nucleotide positions encoding such
different amino acid. The role of each amino acid thus identified as
candidate for backmutation should be investigated for a direct or
indirect role in antigen binding and any amino acid found after mutation
to affect any desirable characteristic of the human antibody should not
be included in the final human antibody; as an example, activity
enhancing amino acids identified by the selective mutagenesis approach
will not be subject to backmutation. To minimize the number of amino
acids subject to backmutation those amino acid positions found to be
different from the closest germline sequence but identical to the
corresponding amino acid in a second germline sequence can remain,
provided that the second germline sequence is identical and colinear to
the sequence of the human antibody of the invention for at least 10,
preferably 12 amino acids, on both sides of the amino acid in question.
Backmutation may occur at any stage of antibody optimization; preferably,
backmutation occurs directly before or after the selective mutagenesis
approach. More preferably, backmutation occurs directly before the
selective mutagenesis approach.

[0310] The phrase "human interleukin 12" (abbreviated herein as hIL-12, or
IL-12), as used herein, includes a human cytokine that is secreted
primarily by macrophages and dendritic cells. The term includes a
heterodimeric protein comprising a 35 kD subunit (p35) and a 40 kD
subunit (p40) which are both linked together with a disulfide bridge. The
heterodimeric protein is referred to as a "p70 subunit". The structure of
human IL-12 is described further in, for example, Kobayashi, et al.
(1989) J. Exp Med. 170:827-845; Seder, et al. (1993) Proc. Natl. Acad.
Sci. 90:10188-10192; Ling, et al. (1995) J. Exp Med. 154:116-127;
Podlaski, et al. (1992) Arch. Biochem. Biophys. 294:230-237. The term
human IL-12 is intended to include recombinant human IL-12 (rh IL-12),
which can be prepared by standard recombinant expression methods.

[0311] The phrase "interleukin comprising a p40 subunit" includes any
interleukin, e.g., any human interleukin, that comprises a p40 subunit.
Such interleukins are well known in the art and include IL-12, e.g.,
human IL-12, and IL-23, e.g., human IL-23.

[0312] The terms "Kabat numbering", "Kabat definitions" and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid residues
which are more variable (i.e. hypervariable) than other amino acid
residues in the heavy and light chain variable regions of an antibody, or
an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad,
Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins
of Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, NIH Publication No. 91-3242). For the heavy chain
variable region, the hypervariable region ranges from amino acid
positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and
amino acid positions 95 to 102 for CDR3. For the light chain variable
region, the hypervariable region ranges from amino acid positions 24 to
34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.

[0313] The Kabat numbering is used herein to indicate the positions of
amino acid modifications made in antibodies of the invention. For
example, the Y61 anti-IL-12 antibody can be mutated from serine (S) to
glutamic acid (E) at position 31 of the heavy chain CDR1 (H31S→E),
or glycine (G) can be mutated to tyrosine (Y) at position 94 of the light
chain CDR3 (L94G→Y).

[0314] The term "human antibody" includes antibodies having variable and
constant regions corresponding to human germline immunoglobulin sequences
as described by Kabat et al. (See Kabat, et al. (1991) Sequences of
proteins of Immunological Interest, Fifth Edition, U.S. Department of
Health and Human Services, NIH Publication No. 91-3242). The human
antibodies of the invention may include amino acid residues not encoded
by human germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation in
vivo), for example in the CDRs and in particular CDR3. The mutations
preferably are introduced using the "selective mutagenesis approach"
described herein. The human antibody can have at least one position
replaced with an amino acid residue, e.g., an activity enhancing amino
acid residue which is not encoded by the human germline immunoglobulin
sequence. The human antibody can have up to twenty positions replaced
with amino acid residues which are not part of the human germline
immunoglobulin sequence. In other embodiments, up to ten, up to five, up
to three or up to two positions are replaced. In a preferred embodiment,
these replacements are within the CDR regions as described in detail
below. However, the term "human antibody", as used herein, is not
intended to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been grafted
onto human framework sequences.

[0315] The phrase "recombinant human antibody" includes human antibodies
that are prepared, expressed, created or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector
transfected into a host cell (described further in Section II, below),
antibodies isolated from a recombinant, combinatorial human antibody
library (described further in Section III, below), antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or isolated
by any other means that involves splicing of human immunoglobulin gene
sequences to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline immunoglobulin
sequences (See Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, NIH Publication No. 91-3242). In certain embodiments,
however, such recombinant human antibodies are subjected to in vitro
mutagenesis (or, when an animal transgenic for human Ig sequences is
used, in vivo somatic mutagenesis) and thus the amino acid sequences of
the VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire in
vivo. In certain embodiments, however, such recombinant antibodies are
the result of selective mutagenesis approach or backmutation or both.

[0316] An "isolated antibody" includes an antibody that is substantially
free of other antibodies having different antigenic specificities (e.g.,
an isolated antibody that specifically binds hIL-12 is substantially free
of antibodies that specifically bind antigens other than hIL-12). An
isolated antibody that specifically binds hIL-12 may bind IL-12 molecules
from other species (discussed in further detail below). Moreover, an
isolated antibody may be substantially free of other cellular material
and/or chemicals.

[0317] A "neutralizing antibody" (or an "antibody that neutralized hIL-12
activity") includes an antibody whose binding to hIL-12 results in
inhibition of the biological activity of hIL-12. This inhibition of the
biological activity of hIL-12 can be assessed by measuring one or more
indicators of hIL-12 biological activity, such as inhibition of human
phytohemagglutinin blast proliferation in a phytohemagglutinin blast
proliferation assay (PHA), or inhibition of receptor binding in a human
IL-12 receptor binding assay (see Example 3-Interferon-gamma Induction
Assay). These indicators of hIL-12 biological activity can be assessed by
one or more of several standard in vitro or in vivo assays known in the
art (see Example 3).

[0318] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-hIL-12 antibody that binds to an IL-12 antigen and/or the
neutralizing potency of an antibody, for example, an anti-hIL-12 antibody
whose binding to hIL-12 inhibits the biological activity of hIL-12, e.g.
inhibition of PHA blast proliferation or inhibition of receptor binding
in a human IL-12 receptor binding assay (see Example 3).

[0320] The term "Koff", as used herein, is intended to refer to the
off rate constant for dissociation of an antibody from the
antibody/antigen complex.

[0321] The term "Kd", as used herein, is intended to refer to the
dissociation constant of a particular antibody-antigen interaction.

[0322] The phrase "nucleic acid molecule" includes DNA molecules and RNA
molecules. A nucleic acid molecule may be single-stranded or
double-stranded, but preferably is double-stranded DNA.

[0323] The phrase "isolated nucleic acid molecule", as used herein in
reference to nucleic acids encoding antibodies or antibody portions
(e.g., VH, VL, CDR3) that bind hIL-12 including "isolated antibodies"),
includes a nucleic acid molecule in which the nucleotide sequences
encoding the antibody or antibody portion are free of other nucleotide
sequences encoding antibodies or antibody portions that bind antigens
other than hIL-12, which other sequences may naturally flank the nucleic
acid in human genomic DNA. Thus, for example, an isolated nucleic acid of
the invention encoding a VH region of an anti-IL-12 antibody contains no
other sequences encoding other VH regions that bind antigens other than
IL-12. The phrase "isolated nucleic acid molecule" is also intended to
include sequences encoding bivalent, bispecific antibodies, such as
diabodies in which VH and VL regions contain no other sequences other
than the sequences of the diabody.

[0324] The term "vector" includes a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One type
of vector is a "plasmid", which refers to a circular double stranded DNA
loop into which additional DNA segments may be ligated. Another type of
vector is a viral vector, wherein additional DNA segments may be ligated
into the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)
can be integrated into the genome of a host cell upon introduction into
the host cell, and thereby are replicated along with the host genome.
Moreover, certain vectors are capable of directing the expression of
genes to which they are operatively linked. Such vectors are referred to
herein as "recombinant expression vectors" (or simply, "expression
vectors"). In general, expression vectors of utility in recombinant DNA
techniques are often in the form of plasmids. In the present
specification, "plasmid" and "vector" may be used interchangeably as the
plasmid is the most commonly used form of vector. However, the invention
is intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions.

[0325] The phrase "recombinant host cell" (or simply "host cell") includes
a cell into which a recombinant expression vector has been introduced. It
should be understood that such terms are intended to refer not only to
the particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the scope of
the term "host cell" as used herein.

[0326] The term "modifying", as used herein, is intended to refer to
changing one or more amino acids in the antibodies or antigen-binding
portions thereof. The change can be produced by adding, substituting or
deleting an amino acid at one or more positions. The change can be
produced using known techniques, such as PCR mutagenesis.

[0327] The phrase "contact position" includes an amino acid position of in
the CDR1, CDR2 or CDR3 of the heavy chain variable region or the light
chain variable region of an antibody which is occupied by an amino acid
that contacts antigen in one of the twenty-six known antibody-antigen
structures. If a CDR amino acid in any of the 26 known solved structures
of antibody-antigen complexes contacts the antigen, then that amino acid
can be considered to occupy a contact position. Contact positions have a
higher probability of being occupied by an amino acid which contact
antigen than non-contact positions. Preferably a contact position is a
CDR position which contains an amino acid that contacts antigen in
greater than 3 of the 26 structures (>11.5%). Most preferably a
contact position is a CDR position which contains an amino acid that
contacts antigen in greater than 8 of the 25 structures (>32%).

[0328] The term "hypermutation position" includes an amino acid residue
that occupies position in the CDR1, CDR2 or CDR3 region of the heavy
chain variable region or the light chain variable region of an antibody
that is considered to have a high frequency or probability for somatic
hypermutation during in vivo affinity maturation of the antibody. "High
frequency or probability for somatic hypermutation" includes frequencies
or probabilities of a 5 to about 40% chance that the residue will undergo
somatic hypermutation during in vivo affinity maturation of the antibody.
It should be understood that all ranges within this stated range are also
intended to be part of this invention, e.g., 5 to about 30%, e.g., 5 to
about 15%, e.g., 15 to about 30%.

[0329] The term "preferred selective mutagenesis position" includes an
amino acid residue that occupies a position in the CDR1, CDR2 or CDR3
region of the heavy chain variable region or the light chain variable
region which can be considered to be both a contact and a hypermutation
position.

[0330] The phrase "selective mutagenesis approach" includes a method of
improving the activity of an antibody by selecting and individually
mutating CDR amino acids at least one preferred selective mutagenesis
position, hypermutation, and/or contact position. A "selectively mutated"
human antibody is an antibody which contains a mutation at a position
selected using a selective mutagenesis approach. In another embodiment,
the selective mutagenesis approach is intended to provide a method of
preferentially mutating selected individual amino acid residues in the
CDR1, CDR2 or CDR3 of the heavy chain variable region (hereinafter H1,
H2, and H3, respectively), or the CDR1, CDR2 or CDR3 of the light chain
variable region (hereinafter referred to as L1, L2, and L3, respectively)
of an antibody. Amino acid residues may be selected from preferred
selective mutagenesis positions, contact positions, or hypermutation
positions. Individual amino acids are selected based on their position in
the light or heavy chain variable region. It should be understood that a
hypermutation position can also be a contact position. In an embodiment,
the selective mutagenesis approach is a "targeted approach". The language
"targeted approach" is intended to include a method of preferentially
mutating selected individual amino acid residues in the CDR1, CDR2 or
CDR3 of the heavy chain variable region or the CDR1, CDR2 or CDR3 of the
light chain variable region of an antibody in a targeted manner, e.g., a
"Group-wise targeted approach" or "CDR-wise targeted approach". In the
"Group-wise targeted approach", individual amino acid residues in
particular groups are targeted for selective mutations including groups I
(including L3 and H3), II (including H2 and L1) and III (including L2 and
H1), the groups being listed in order of preference for targeting. In the
"CDR-wise targeted approach", individual amino acid residues in
particular CDRs are targeted for selective mutations with the order of
preference for targeting as follows: H3, L3, H2, L1, H1 and L2. The
selected amino acid residue is mutated, e.g., to at least two other amino
acid residues, and the effect of the mutation on the activity of the
antibody is determined. Activity is measured as a change in the binding
specificity/affinity of the antibody, and/or neutralization potency of
the antibody. It should be understood that the selective mutagenesis
approach can be used for the optimization of any antibody derived from
any source including phage display, transgenic animals with human IgG
germline genes, human antibodies isolated from human B-cells. Preferably,
the selective mutagenesis approach is used on antibodies which can not be
optimized further using phage display technology. It should be understood
that antibodies from any source including phage display, transgenic
animals with human IgG germline genes, human antibodies isolated from
human B-cells can be subject to backmutation prior to or after the
selective mutagenesis approach.

[0331] The term "activity enhancing amino acid residue" includes an amino
acid residue which improves the activity of the antibody. It should be
understood that the activity enhancing amino acid residue may replace an
amino acid residue at a preferred selective mutagenesis position, contact
position, or a hypermutation position and, further, more than one
activity enhancing amino acid residue can be present within one or more
CDRs. An activity enchancing amino acid residue include, an amino acid
residue that improves the binding specificity/affinity of an antibody,
for example anti-human IL-12 antibody binding to human IL-12. The
activity enhancing amino acid residue is also intended to include an
amino acid residue that improves the neutralization potency of an
antibody, for example, the human IL-12 antibody which inhibits human
IL-12.

[0332] Various aspects of the invention are described in further detail in
the following subsections.

I. Human Antibodies that Bind Human IL-12

[0333] This invention provides isolated human antibodies, or
antigen-binding portions thereof, that bind to human IL-12. Preferably,
the human antibodies of the invention are recombinant, neutralizing human
anti-hIL-12 antibodies. Antibodies of the invention that bind to human
IL-12 can be selected, for example, by screening one or more human VL and
VH cDNA libraries with hIL-12, such as by phage display techniques as
described in Example 1. Screening of human VL and VH cDNA libraries
initially identified a series of anti-IL-12 antibodies of which one
antibody, referred to herein as "Joe 9" (or "Joe 9 wild type"), was
selected for further development. Joe 9 is a relatively low affinity
human IL-12 antibody (e.g., a Koff of about 0.1 sec-1), yet is
useful for specifically binding and detecting hIL-12. The affinity of the
Joe 9 antibody was improved by conducting mutagenesis of the heavy and
light chain CDRs, producing a panel of light and heavy chain variable
regions that were "mixed and matched" and further mutated, leading to
numerous additional anti-hIL-12 antibodies with increased affinity for
hIL-12 (see Example 1, Table 2 (see Appendix A) and the sequence
alignments of FIGS. 1A-D).

[0334] Of these antibodies, the human anti-hIL-12 antibody referred to
herein as Y61 demonstrated a significant improvement in binding affinity
(e.g., a Koff of about 2×10-4 sec-1). The Y61
anti-hIL-12 antibody was selected for further affinity maturation by
individually mutating specific amino acids residues within the heavy and
light chain CDRs. Amino acids residues of Y61 were selected for
site-specific mutation (selective mutagenesis approach) based on the
amino acid residue occupying a preferred selective mutagenesis position,
contact and/or a hypermutation position. A summary of the substitutions
at selected positions in the heavy and light chain CDRs is shown in FIGS.
2A-2H. A preferred recombinant neutralizing antibody of the invention,
referred to herein as J695, resulted from a Gly to Tyr substitution at
position 50 of the light chain CDR2 of Y61, and a Gly to Tyr substitution
at position 94 of the light chain CDR3 of Y61.

[0336] Antibodies produced from affinity maturation of Joe 9 wild type
were functionally characterized by surface plasmon resonance analysis to
determine the Kd and Koff rate. A series of antibodies were
produced having a Koff rate within the range of about 0.1 s-1
to about 1×10-5 s-1, and more preferably a Koff of
about 1×10-4 s-1 to 1×10-5 s-1 or less.
Antibodies were also characterized in vitro for their ability to inhibit
phytohemagglutinin (PHA) blast proliferation, as described in Example 3.
A series of antibodies were produced having an IC50 value in the
range of about 1×10-6 M to about 1×10-11 M, more
preferably about 1×10-10 M to 1×10-11 M or less.

[0337] Accordingly, in one aspect, the invention provides an isolated
human antibody, or antigen-binding portion thereof, that binds to human
IL-12 and dissociates from human IL-12 with a Koff rate constant of
0.1 s-1 or less, as determined by surface plasmon resonance, or
which inhibits phytohemagglutinin blast proliferation in an in vitro
phytohemagglutinin blast proliferation assay (PHA assay) with an
IC50 of 1×10-6 M or less. In preferred embodiments, the
isolated human IL-12 antibody, or an antigen-binding portion thereof,
dissociates from human IL-12 with a Koff rate constant of
1×10-2 s-1 or less, or inhibits phytohemagglutinin blast
proliferation in an in vitro PHA assay with an IC50 of
1×10-7 M or less. In more preferred embodiments, the isolated
human IL-12 antibody, or an antigen-binding portion thereof, dissociates
from human IL-12 with a Koff rate constant of 1×10-3
s-1 or less, or inhibits phytohemagglutinin blast proliferation in
an in vitro PHA assay with an IC50 of 1×10-8 M or less.
In more preferred embodiments, the isolated human IL-12 antibody, or an
antigen-binding portion thereof, dissociates from human IL-12 with a
Koff rate constant of 1×10-4 s-1 or less, or
inhibits phytohemagglutinin blast proliferation in an in vitro PHA assay
with an IC50 of 1×10-9 M or less. In more preferred
embodiments, the isolated human IL-12 antibody, or an antigen-binding
portion thereof, dissociates from human IL-12 with a Koff rate
constant of 1×10-5 s-1 or less, or inhibits
phytohemagglutinin blast proliferation in an in vitro PHA assay with an
IC50 of 1×10-10 M or less. In even more preferred
embodiments, the isolated human IL-12 antibody, or an antigen-binding
portion thereof, dissociates from human IL-12 with a Koff rate
constant of 1×10-5 s-1 or less, or inhibits
phytohemagglutinin blast proliferation in an in vitro PHA assay with an
IC50 of 1×10-11 M or less.

[0338] The dissociation rate constant (Koff) of an IL-12 antibody can
be determined by surface plasmon resonance (see Example 5). Generally,
surface plasmon resonance analysis measures real-time binding
interactions between ligand (recombinant human IL-12 immobilized on a
biosensor matrix) and analyte (antibodies in solution) by surface plasmon
resonance (SPR) using the BIAcore system (Pharmacia Biosensor,
Piscataway, N.J.). Surface plasmon analysis can also be performed by
immobilizing the analyte (antibodies on a biosensor matrix) and
presenting the ligand (recombinant IL-12 in solution). Neutralization
activity of IL-12 antibodies, or antigen binding portions thereof, can be
assessed using one or more of several suitable in vitro assays (see
Example 3).

[0339] It is well known in the art that antibody heavy and light chain
CDRs play an important role in the binding specificity/affinity of an
antibody for an antigen. Accordingly, the invention encompasses human
antibodies having light and heavy chain CDRs of Joe 9, as well as other
antibodies having CDRs that have been modified to improve the binding
specificity/affinity of the antibody. As demonstrated in Example 1, a
series of modifications to the light and heavy chain CDRs results in
affinity maturation of human anti-hIL-12 antibodies. The heavy and light
chain variable region amino acid sequence alignments of a series of human
antibodies ranging from Joe 9 wild type to J695 that bind human IL-12 is
shown in FIGS. 1A-1D. Consensus sequence motifs for the CDRs of
antibodies can be determined from the sequence alignment (as summarized
in the table above). For example, a consensus motif for the VH CDR3 of
the lineage from Joe 9 to J695 comprises the amino acid sequence:
(H/S)-G-S-(H/Y)-D-(N/T/Y) (SEQ ID NO: 1), which encompasses amino acids
from position 95 to 102 of the consensus HCVR shown in SEQ ID NO: 7. A
consensus motif for the VL CDR3 comprises the amino acid sequence:
Q-(S/T)-Y-(D/E)-(S/R/K)-(S/G/Y)-(L/F/T/S)-(R/S/T/W/H)-(G/P)-(S/T/A/L)-(R/-
S/M/T/L-V/I/T/M/L) (SEQ ID NO: 2), which encompasses amino acids from
position 89 to 97 of the consensus LCVR shown in SEQ ID NO: 8.

[0340] Accordingly, in another aspect, the invention provides an isolated
human antibody, or an antigen-binding portion thereof, which has the
following characteristics:

[0341] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-6 M or less;

[0346] In yet another preferred embodiment, the antibody of the invention
comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 7 and a
LCVR comprising the amino acid sequence of SEQ ID NO: 8.

[0347] Additional consensus motifs can be determined based on the
mutational analysis performed on Y61 that led to the J695 antibody
(summarized in FIGS. 2A-2H). As demonstrated by the graphs shown in FIGS.
2A-2H, certain residues of the heavy and light chain CDRs of Y61 were
amenable to substitution without significantly impairing the hIL-12
binding properties of the antibody. For example, individual substitutions
at position 30 in CDR H1 with twelve different amino acid residues did
not significantly reduce the Koff rate of the antibody, indicating
that is position is amenable to substitution with a variety of different
amino acid residues. Thus, based on the mutational analysis (i.e.,
positions within Y61 that were amenable to substitution by other amino
acid residues) consensus motifs were determined. The consensus motifs for
the heavy and light chain CDR3s are shown in SEQ ID NOs: 9 and 10,
respectively, consensus motifs for the heavy and light chain CDR2s are
shown in SEQ ID NOs: 11 and 12, respectively, and consensus motifs for
the heavy and light chain CDR1s are shown in SEQ ID NOs: 13 and 14,
respectively. Consensus motifs for the VH and VL regions are shown in SEQ
ID NOs: 15 and 16, respectively.

[0348] Accordingly, in one aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which has the
following characteristics:

[0349] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0352] In a preferred embodiment, the antibody further comprises a VH CDR2
comprising the amino acid sequence of SEQ ID NO: 11 and further comprises
a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 12.

[0353] In another preferred embodiment, the antibody further comprises a
VH CDR1 comprising the amino acid sequence of SEQ ID NO: 13 and further
comprises a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0354] In yet another preferred embodiment, the antibody of the invention
comprises a HCVR comprising the amino acid sequence of SEQ ID NO: 15 and
a LCVR comprising the amino acid sequence of SEQ ID NO: 16.

[0355] A preferred antibody of the invention, the human anti-hIL-12
antibody Y61, was produced by affinity maturation of Joe 9 wild type by
PCR mutagenesis of the CDR3 (as described in Example 1). Y61 had an
improved specificity/binding affinity determined by surface plasmon
resonance and by in vitro neutralization assays. The heavy and light
chain CDR3s of Y61 are shown in SEQ ID NOs: 17 and 18, respectively, the
heavy and light chain CDR2s of Y61 are shown in SEQ ID NOs: 19 and 20,
respectively, and the heavy and light chain CDR1s of Y61 are shown in SEQ
ID NOs: 21 and 22, respectively. The VH of Y61 has the amino acid
sequence of SEQ ID NO: 23 and the VL of Y61 has the amino acid sequence
of SEQ ID NO: 24 (these sequences are also shown in FIGS. 1A-1D, aligned
with Joe9).

[0356] Accordingly, in another aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which

[0357] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0363] In certain embodiments, the full length antibody comprises a heavy
chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE
constant regions, and any allotypic variant therein as described in Kabat
(Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services,
NIH Publication No. 91-3242). Preferably, the antibody heavy chain
constant region is an IgG1 heavy chain constant region. Alternatively,
the antibody portion can be an Fab fragment, an F(ab'2) fragment or
a single chain Fv fragment.

[0364] Modifications of individual residues of Y61 led to the production
of a panel of antibodies shown in FIGS. 2A-2H. The specificity/binding
affinity of each antibody was determined by surface plasmon resonance
and/or by in vitro neutralization assays.

[0365] Accordingly, in another aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which

[0366] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0367] b) has a heavy chain CDR3 comprising the amino acid sequence
selected from the group consisting of SEQ ID NO: 404-SEQ ID NO: 469; and

[0372] In certain embodiments, the full length antibody comprising a heavy
chain constant region such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE
constant regions and any allotypic variant therein as described in Kabat
(Kabat, E. A., et al. (1991) Sequences of Proteins of immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services,
NIH Publication No. 91-3242). Preferably, the antibody heavy chain
constant region is an IgG1 heavy chain constant region. Alternatively,
the antibody portion can be a Fab fragment, an F(ab'2) fragment or a
single chain Fv fragment.

[0373] A particularly preferred recombinant, neutralizing antibody of the
invention, J695, was produced by site-directed mutagenesis of contact and
hypermutation amino acids residues of antibody Y61 (see Example 2 and
section III below). J695 differs from Y61 by a Gly to Tyr substitution in
Y61 at position 50 of the light chain CDR2 and by a Gly to Tyr
substitution at position 94 of the light chain CDR3. The heavy and light
chain CDR3s of J695 are shown in SEQ ID NOs: 25 and 26, respectively, the
heavy and light chain CDR2s of J695 are shown in SEQ ID NOs: 27 and 28,
respectively, and the heavy and light chain CDR1s of J695 are shown in
SEQ ID NOs: 29 and 30, respectively. The VH of J695 has the amino acid
sequence of SEQ ID NO: 31 and the VL of J695 has the amino acid sequence
of SEQ ID NO: 32 (these sequences are also shown in FIGS. 1A-1D, aligned
with Joe9).

[0374] Accordingly, in another aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which

[0375] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0381] In certain embodiments, the full length antibody comprises a heavy
chain constant region, such as IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE
constant regions and any allotypic variant therein as described in Kabat
(Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services,
NIH Publication No. 91-3242). Preferably, the antibody heavy chain
constant region is an IgG1 heavy chain constant region. Alternatively,
the antibody portion can be an Fab fragment, an F(ab'2) fragment or
a single chain Fv fragment.

[0382] Additional mutations in the preferred consensus sequences for CDR3,
CDR2, and CDR1 of antibodies on the lineage from Joe 9 to J695, or from
the lineage Y61 to J695, can be made to provide additional anti-IL-12
antibodies of the invention. Such methods of modification can be
performed using standard molecular biology techniques, such as by PCR
mutagenesis, targeting individual contact or hypermutation amino acid
residues in the light chain and/or heavy chain CDRs-, followed by kinetic
and functional analysis of the modified antibodies as described herein
(e.g., neutralization assays described in Example 3, and by BIAcore
analysis, as described in Example 5).

[0383] Accordingly, in another aspect the invention features an isolated
human antibody, or an antigen-binding portion thereof, which

[0384] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-6 M or less;

[0385] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 3 and a heavy chain CDR1 comprising the amino acid sequence of
SEQ ID NO: 5, or a mutant thereof having one or more amino acid
substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 5; and

[0386] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 2, a light chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 4, and a light chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 6, or a mutant thereof having one or more amino acid
substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 2, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 4, and a light chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 6.

[0387] In another aspect the invention features an isolated human
antibody, or an antigen-binding portion thereof, which

[0388] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0389] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of
SEQ ID NO: 11 and a heavy chain CDR1 comprising the amino acid sequence
of SEQ ID NO: 13, or a mutant thereof having one or more amino acid
substitutions at a preferred selective mutagenesis position, contact
position or a hypermutation position, wherein said mutant has a koff
rate no more than 10-fold higher than the antibody comprising a heavy
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9, a heavy
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a
heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13; and

[0390] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 10, a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 12, and a light chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 14, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position, contact
position or a hypermutation position, wherein said mutant has a koff
rate no more than 10-fold higher than the antibody comprising a light
chain CDR3 comprising the amino acid sequence of SEQ ID NO: 10, a light
chain CDR2 comprising the amino acid sequence of SEQ ID NO: 12, and a
light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 14.

[0391] An ordinarily skilled artisan will also appreciate that additional
mutations to the CDR regions of an antibody of the invention, for example
in Y61 or in J695, can be made to provide additional anti-IL-12
antibodies of the invention. Such methods of modification can be
performed using standard molecular biology techniques, as described
above. The functional and kinetic analysis of the modified antibodies can
be performed as described in Example 3 and Example 5, respectively.
Modifications of individual residues of Y61 that led to the
identification of J695 are shown in FIGS. 2A-2H and are described in
Example 2.

[0392] Accordingly, in another aspect the invention features an isolated
human antibody, or an antigen-binding portion thereof, which

[0393] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0394] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 19 and a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 21, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 19, and a heavy chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 21; and

[0395] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 18, a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 20, and a light chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 22, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 18, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 20, and a light chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 22.

[0396] In another aspect the invention features an isolated human
antibody, or an antigen-binding portion thereof, which

[0397] a) inhibits phytohemagglutinin blast proliferation in an in vitro
PHA assay with an IC50 of 1×10-9 M or less;

[0398] b) comprises a heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 27 and a heavy chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 29, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a heavy chain CDR3
comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2
comprising the amino acid sequence of SEQ ID NO: 27, and a heavy chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 29; and

[0399] c) comprises a light chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence
of SEQ ID NO: 28, and a light chain CDR1 comprising the amino acid
sequence of SEQ ID NO: 30, or a mutant thereof having one or more amino
acid substitutions at a preferred selective mutagenesis position or a
hypermutation position, wherein said mutant has a koff rate no more
than 10-fold higher than the antibody comprising a light chain CDR3
comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2
comprising the amino acid sequence of SEQ ID NO: 28, and a light chain
CDR1 comprising the amino acid sequence of SEQ ID NO: 30.

[0400] In yet another embodiment, the invention provides isolated human
antibodies, or antigen-binding portions thereof, that neutralize the
activity of human IL-12, and at least one additional primate IL-12
selected from the group consisting of baboon IL-12, marmoset IL-12,
chimpanzee IL-12, cynomolgus IL-12 and rhesus IL-12, but which do not
neutralize the activity of the mouse IL-12.

[0402] The antibody libraries used in this method are preferably scFv
libraries prepared from human VL and VH cDNAs. The scFv antibody
libraries are preferably screened using recombinant human IL-12 as the
antigen to select human heavy and light chain sequences having a binding
activity toward IL-12. To select for antibodies specific for the p35
subunit of IL-12 or the p70 heterodimer, screening assays were performed
in the presence of excess free p40 subunit. Subunit preferences can be
determined, for example by, micro-Friguet titration, as described in
Example 1.

[0403] Once initial human VL and VH segments are selected, "mix and match"
experiments, in which different pairs of the selected VL and VH segments
are screened for IL-12 binding, are performed to select preferred VL/VH
pair combinations (see Example 1). Additionally, to further improve the
affinity and/or lower the off rate constant for hIL-12 binding, the VL
and VH segments of the preferred VL/VH pair(s) can be randomly mutated,
preferably within the CDR3 region of VH and/or VL, in a process analogous
to the in vivo somatic mutation process responsible for affinity
maturation of antibodies during a natural immune response. This in vitro
affinity maturation can be accomplished by amplifying VH and VL regions
using PCR primers complimentary to the VH CDR3 or VL CDR3, respectively,
which primers have been "spiked" with a random mixture of the four
nucleotide bases at certain positions such that the resultant PCR
products encode VH and VL segments into which random mutations have been
introduced into the VH and/or VL CDR3 regions. These randomly mutated VH
and VL segments can be reselected and rescreened for binding to hIL-12
and sequences that exhibit high affinity and a low off rate for IL-12
binding can be selected. Table 2 (see Appendix A) shows antibodies that
displayed altered binding specificity/affinity produced as a result of in
vitro affinity maturation.

[0404] Following selection, isolation and screening of an anti-hIL-12
antibody of the invention from a recombinant immunoglobulin display
library, nucleic acid encoding the selected antibody can be recovered
from the phage particle(s) (e.g. from the phage genome) and subcloned
into other expression vectors by standard recombinant DNA techniques. If
desired, the nucleic acid can be further manipulated to create other
antibody forms of the invention (e.g., linked to nucleic acid encoding
additional immunoglobulin domains, such as additional constant regions).
To express a recombinant human antibody isolated by screening of a
combinatorial library, the DNA encoding the antibody is cloned into a
recombinant expression vector and introduced into a mammalian host cells,
as described in further detail in Section IV below.

[0405] Methods for selecting human IL-12 binding antibodies by phage
display technology, and affinity maturation of selected antibodies by
random or site-directed mutagenesis of CDR regions are described in
further detail in Example 1.

[0406] As described in Example 1, screening of human VL and VH cDNA
libraries identified a series of anti-IL-12 antibodies, of which the Joe
9 antibody was selected for further development. A comparison of the
heavy chain variable region of Joe 9 with the heavy chain germline
sequences selected from the VBASE database, revealed that Joe 9 was
similar to the COS-3 germline sequence. COS-3 belongs to the VH3
family of germline sequences.

[0407] The VH3 family is part of the human VH germline repertoire
which is grouped into seven families, V.sub.H1-V.sub.H7, based on
nucleotide sequence homology (Tomlinson et al. (1992) J. Mol. Biol., 227,
776-798 and Cook et al. (1995) Immunology Today, 16, 237-242). The
VH3 family contains the highest number of members and makes the
largest contribution to the germline repertoire. For any given human
VH3-germline antibody sequence, the amino acid sequence identity
within the entire VH3 family is high (See e.g., Tomlinson et al.
(1992) J. Mol. Biol., 227, 776-798 and Cook et al. (1995) Immunology
Today, 16, 237-242). The range of amino acid sequence identity between
any two germline VH sequences of the VH3 family varies from 69-98
residues out of approximately 100 VH residues, (i.e., 69-98% amino acid
sequence homology between any two germline VH sequences). For most pairs
of germline sequences there is at least 80 or more identical amino acid
residues, (i.e., at least 80% amino acid sequence homology). The high
degree of amino acid sequence homology between the VH3 family
members results in certain amino acid residues being present at key sites
in the CDR and framework regions of the VH chain. These amino acid
residues confer structural features upon the CDRs.

[0408] Studies of antibody structures have shown that CDR conformations
can be grouped into families of canonical CDR structures based on the key
amino acid residues that occupy certain positions in the CDR and
framework regions. Consequently, there are similar local CDR
conformations in different antibodies that have canonical structures with
identical key amino acid residues (Chothia et al. (1987) J. Mol. Biol.,
196, 901-917 and Chothia et al. (1989) Nature, 342, 877-883). Within the
VH3 family there is a conservation of amino acid residue identity at
the key sites for the CDR1 and CDR2 canonical structures (Chothia et al.
(1992) J. Mol. Biol., 227, 799-817).

[0409] The COS-3 germline VH gene, is a member of the VH3 family and
is a variant of the 3-30 (DP-49) germline VH allele. COS-3, differs from
Joe9 VH amino acid sequences at only 5 positions. The high degree of
amino acid sequence homology between Joe9 VH and COS-3, and between Joe9
VH and the other VH3 family members also confers a high degree of
CDR structural homology (Chothia et al. (1992) J. Mol. Biol., 227,
799-817; Chothia et al. (1987) J. Mol. Biol., 196, 901-917 and Chothia et
al. (1989) Nature, 342, 877-883).

[0410] The skilled artisan will appreciate that based on the high amino
acid sequence and canonical structural similarity to Joe 9, other
VH3 family members could also be used to generate antibodies that
bind to human IL-12. This can be performed, for example, by selecting an
appropriate VL by chain-shuffling techniques (Winter et al. (1994) Annual
Rev. Immunol., 12, 433-55), or by the grafting of CDRs from a rodent or
other human antibody including CDRs from antibodies of this invention
onto a VH3 family framework.

[0411] The human V lambda germline repertoire is grouped into 10 families
based on nucleotide sequence homology (Williams et al. (1996) J. Mol.
Biol., 264, 220-232). A comparison of the light chain variable region of
Joe 9 with the light chain germline sequences selected from the VBASE
database, revealed that Joe 9 was similar to the DPL8 lambda germline.
The Joe9 VL differs from DPL8 sequence at only four framework positions,
and is highly homologous to the framework sequences of the other
V.sub.λ1 family members. Based on the high amino acid sequence
homology and canonical structural similarity to Joe 9, other
V.sub.λ1 family members may also be used to generate antibodies
that bind to human IL-12. This can be performed, for example, by
selecting an appropriate VH by chain-shuffling techniques (Winter et al.
Supra, or by the grafting of CDRs from a rodent or other human antibody
including CDRs from antibodies of this invention onto a V.sub.λ1
family framework.

[0412] The methods of the invention are intended to include recombinant
antibodies that bind to hIL-12, comprising a heavy chain variable region
derived from a member of the VH3 family of germline sequences, and a
light chain variable region derived from a member of the V.sub.λ1
family of germline sequences. Moreover, the skilled artisan will
appreciate that any member of the VH3 family heavy chain sequence
can be combined with any member of the V.sub.λ1 family light chain
sequence.

[0413] Those skilled in the art will also appreciate that DNA sequence
polymorphisms that lead to changes in the amino acid sequences of the
germline may exist within a population (e.g., the human population). Such
genetic polymorphism in the germline sequences may exist among
individuals within a population due to natural allelic variation. Such
natural allelic variations can typically result in 1-5% variance in the
nucleotide sequence of the a gene. Any and all such nucleotide variations
and resulting amino acid polymorphisms in germline sequences that are the
result of natural allelic variation are intended to be within the scope
of the invention.

[0414] Accordingly, in one aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which has the
following characteristics: [0415] a) that binds to human IL-12 and
dissociates from human IL-12 with a koff rate constant of 0.1
s-1 or less, as determined by surface plasmon resonance, or which
inhibits phytohemagglutinin blast proliferation in an in vitro
phytohemagglutinin blast proliferation assay (PHA assay) with an
IC50 of 1×10-6M or less. [0416] b) has a heavy chain
variable region comprising an amino acid sequence selected from a member
of the VH3 germline family, wherein the heavy chain variable region
has a mutation at a contact or hypermutation position with an activity
enhancing amino acid residue. [0417] c) has a light chain variable
region comprising an amino acid sequence selected from a member of the
V.sub.λ1 germline family, wherein the light chain variable region
has a mutation at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid residue.

[0418] In a preferred embodiment, the isolated human antibody, or antigen
binding has mutation in the heavy chain CDR3.

[0419] In another preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the light chain CDR3.

[0420] In another preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the heavy chain CDR2.

[0421] In another preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the light chain CDR2.

[0422] In another preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the heavy chain CDR1.

[0423] In another preferred embodiment, the isolated human antibody, or
antigen binding has mutation in the light chain CDR1.

[0424] An ordinarily skilled artisan will appreciate that based on the
high amino acid sequence similarity between members of the VH3
germline family, or between members of the light chain V.sub.λ1
germline family, that mutations to the germlines sequences can provide
additional antibodies that bind to human IL-12. Table 1 (see Appendix A)
shows the germline sequences of the VH3 family members and
demonstrates the significant sequence homology within the family members.
Also shown in Table 1 are the germline sequences for V.sub.λ1
family members. The heavy and light chain sequences of Joe 9 are provided
as a comparison. Mutations to the germline sequences of VH3 or
V.sub.λ1 family members may be made, for example, at the same amino
acid positions as those made in the antibodies of the invention (e.g.
mutations in Joe 9). The modifications can be performed using standard
molecular biology techniques, such as by PCR mutagenesis, targeting
individual amino acid residues in the germline sequences, followed by
kinetic and functional analysis of the modified antibodies as described
herein (e.g., neutralization assays described in Example 3, and by
BIAcore analysis, as described in Example 5).

[0425] Accordingly, in one aspect, the invention features isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0426] a) has a heavy chain variable region
comprising an amino acid sequence selected from the group consisting of
SEQ ID NOs: 595-667, wherein the heavy chain variable region has a
mutation at a preferred selective mutagenesis position, contact or
hypermutation position with an activity enhancing amino acid residue.
[0427] b) has a light chain variable region comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 669-675,
wherein the light chain variable region has a mutation at a preferred
selective mutagenesis position, contact or hypermutation position with an
activity enhancing amino acid residue.

[0428] An ordinarily skilled artisan will appreciate that based on the
high amino acid sequence similarity between Joe 9 and COS-3 heavy chain
germline sequence, and between Joe 9 and DPL8 lambda germline sequence,
that other mutations to the CDR regions of these germlines sequences can
provide additional antibodies that bind to human IL-12. Such methods of
modification can be performed using standard molecular biology techniques
as described above.

[0429] Accordingly, in one aspect, the invention features isolated human
antibody, or an antigen-binding portion thereof, which has the following
characteristics: [0430] a) that binds to human IL-12 and dissociates
from human IL-12 with a koff rate constant of 0.1 s-1 or less,
as determined by surface plasmon resonance, or which inhibits
phytohemagglutinin blast proliferation in an in vitro phytohemagglutinin
blast proliferation assay (PHA assay) with an IC50 of
1×10-6M or less. [0431] b) has a heavy chain variable region
comprising the COS-3 germline amino acid sequence, wherein the heavy
chain variable region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity enhancing
amino acid residue. [0432] c) has a light chain variable region
comprising the DPL8 germline amino acid sequence, wherein the light chain
variable region has a mutation at a preferred selective mutagenesis
position, contact or hypermutation position with an activity enhancing
amino acid residue.

[0433] Due to certain amino acid residues occupying key sites in the CDR
and framework regions in the light and heavy chain variable region,
structural features are conferred at these regions. In particular, the
CDR2 and CDR1 regions are subject to canonical structural
classifications. Since there is a high degree of amino acids sequence
homology between family members, these canonical features are present
between family members. The skilled artisan will appreciate that
modifications at the amino acid residues that confer these canonical
structures would produce additional antibodies that bind to IL-12. The
modifications can be performed using standard molecular biology
techniques as described above.

[0434] Accordingly, in another aspect, the invention features an isolated
human antibody, or an antigen-binding portion thereof, which has the
following characteristics: [0435] a) that binds to human IL-12 and
dissociates from human IL-12 with a koff rate constant of 0.1
s-1 or less, as determined by surface plasmon resonance, or which
inhibits phytohemagglutinin blast proliferation in an in vitro
phytohemagglutinin blast proliferation assay (PHA assay) with an
IC50 of 1×10-6M or less. [0436] b) has a heavy chain
variable region comprising an amino acid sequence selected from a member
of the VH3 germline family, wherein the heavy chain variable region
comprises a CDR2 that is structurally similar to CDR2s from other
VH3 germline family members, and a CDR1 that is structurally similar
to CDR1s from other VH3 germline family members, and wherein the
heavy chain variable region has a mutation at a preferred selective
mutagenesis position, contact or hypermutation position with an activity
enhancing amino acid residue; [0437] c) has a light chain variable
region comprising an amino acid sequence selected from a member of the
V.sub.λ1 germline family, wherein the light chain variable region
comprises a CDR2 that is structurally similar to CDR2s from other
V.sub.λ1 germline family members, and a CDR1 that is structurally
similar to CDR1s from other V.sub.λ1 germline family members, and
wherein the light chain variable region has a mutation at a preferred
selective mutagenesis position, contact or hypermutation position with an
activity enhancing amino acid residue.

[0438] Recombinant human antibodies of the invention have variable and
constant regions which are homologous to human germline immunoglobulin
sequences selected from the VBASE database. Mutations to the recombinant
human antibodies (e.g., by random mutagenesis or PCR mutagenesis) result
in amino acids that are not encoded by human germline immunoglobulin
sequences. Also, libraries of recombinant antibodies which were derived
from human donors will contain antibody sequences that differ from their
corresponding germline sequences due to the normal process of somatic
mutation that occurs during B-cell development. It should be noted that
if the "germline" sequences obtained by PCR amplification encode amino
acid differences in the framework regions from the true germline
configuration (i.e., differences in the amplified sequence as compared to
the true germline sequence), it may be desirable to change these amino
acid differences back to the true germline sequences (i.e.,
"backmutation" of framework residues to the germline configuration).
Thus, the present invention can optionally include a backmutation step.
To do this, the amino acid sequences of heavy and light chain encoded by
the germline (as found as example in VBASE database) are first compared
to the mutated immunoglobulin heavy and light chain framework amino acid
sequences to identify amino acid residues in the mutated immunoglobulin
framework sequence that differ from the closest germline sequences. Then,
the appropriate nucleotides of the mutated immunoglobulin sequence are
mutated back to correspond to the germline sequence, using the genetic
code to determine which nucleotide changes should be made. Mutagenesis of
the mutated immunoglobulin framework sequence is carried out by standard
methods, such as PCR-mediated mutagenesis (in which the mutated
nucleotides are incorporated into the PCR primers such that the PCR
product contains the mutations) or site-directed mutagenesis. The role of
each amino acid identified as candidate for backmutation should be
investigated for a direct or indirect role in antigen binding and any
amino acid found after mutation to affect any desirable characteristic of
the human antibody should not be included in the final human antibody; as
an example, activity enhancing amino acids identified by the selective
mutagenesis approach will not be subject to backmutation. Assays to
determine the characteristics of the antibody resulting from mutagenesis
can include ELISA, competitive ELISA, in vitro and in vivo neutralization
assays and/or (see e.g. Example 3) immunohistochemistry with tissue
sections from various sources (including human, primate and/or other
species).

[0439] To minimize the number of amino acids subject to backmutation those
amino acid positions found to be different from the closest germline
sequence but identical to the corresponding amino acid in a second
germline sequence can remain, provided that the second germline sequence
is identical and colinear to the sequence of the human antibody of the
invention for at least 10, preferably 12 amino acids, on both sides of
the amino acid in question. This would assure that any peptide epitope
presented to the immune system by professional antigen presenting cells
in a subject treated with the human antibody of the invention would not
be foreign but identical to a self-antigen, i.e. the immunoglobulin
encoded by that second germline sequence. Backmutation may occur at any
stage of antibody optimization; preferably, backmutation occurs directly
before or after the selective mutagenesis approach. More preferably,
backmutation occurs directly before the selective mutagenesis approach.

[0440] Typically, selection of antibodies with improved affinities can be
carried out using phage display methods, as described in section II
above. This can be accomplished by randomly mutating combinations of CDR
residues and generating large libraries containing antibodies of
different sequences. However, for these selection methods to work, the
antibody-antigen reaction must tend to equilibrium to allow, over time,
preferential binding of higher affinity antibodies to the antigen.
Selection conditions that would allow equilibrium to be established could
not be determined (presumably due to additional non-specific interactions
between the antigen and phage particle) when phage display methods were
used to improve the affinity of selected anti-IL-12 antibodies, upon
attaining a certain level of affinity achieved (i.e., that of antibody
Y61). Accordingly, antibodies with even higher affinities could not be
selected by phage display methods. Thus, for at least certain antibodies
or antigens, phage display methods are limiting in their ability to
select antibodies with a highly improved binding specificity/affinity.
Accordingly, a method termed Selective Mutagenesis Approach which does
not require phage display affinity maturation of antibodies, was
established to overcome this limitation and is provided by the invention.
Although this Selective Mutagenesis Approach was developed to overcome
limitations using the phage display system, it should be noted that this
method can also be used with the phage display system. Moreover, the
selective mutagenesis approach can be used to improve the activity of any
antibody.

[0441] To improve the activity (e.g., affinity or neutralizing activity)
of an antibody, ideally one would like to mutate every CDR position in
both the heavy and light chains to every other possible amino acid
residue. However, since there are, on average, 70 CDR positions within an
antibody, such an approach would be very time consuming and labor
intensive. Accordingly, the method of the invention allows one to improve
the activity of the antibody by mutating only certain selected residues
within the heavy and/or light chain CDRs. Furthermore, the method of the
invention allows improvement in activity of the antibody without
affecting other desirable properties of the antibody.

[0442] Determining which amino acid residues of an antibody variable
region are in contact with an antigen cannot be accurately predicted
based on primary sequence or their positions within the variable region.
Nevertheless, alignments of sequences from antibodies with different
specificities conducted by Kabat et al. have identified the CDRs as local
regions within the variable regions which differ significantly among
antibodies (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-393, Kabat, E.
A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH Publication
No. 91-3242). Structural studies have shown that the antigen binding
surface is formed by amino acid residues present in the CDRs. Other amino
acid residues outside the CDR are also known to play structural roles or
be directly involved in antigen binding. Therefore, for each
antigen-antibody pair, amino acid residues within and outside of the CDRs
may be important.

[0443] The sequence alignment studies by Tomlison et al identified a
number of positions in the heavy and light chain CDR1 and CDR2, and in a
portion of the kappa chain CDR3 which are frequent sites of somatic
mutation. (Tomlison et al (1996) J. Mol. Biol. 256: 813-817). In
particular, positions H31, H31B, H33, H33B, H52B, H56, H58, L30,
L31, L31A, L50, L53, L91, L92, L93 and L94 were identified as frequent
sites for somatic mutation. However, this analysis excludes the important
heavy chain CDR3 regions, and sections of the light chain CDR3 which are
known to lie in the center of an antibody binding site, and potentially
provide important interactions with an antigen. Furthermore, Tomlison et
al. propose that somatic diversity alone does not necessarily predict a
role of a specific amino acid in antigen binding, and suggest conserved
amino acid residues that contact the antigen, and diverse amino acid
residues which do not contact the antigen. This conclusion is further
supported by mutational studies on the role of somatic mutations to
antibody affinity (Sharon, (1990), PNAS, 87:4814-7). Nineteen somatic
mutations in a high-affinity anti-p-azophenylarsonate (Ars) antibody were
simultaneously replaced with their corresponding germline residues,
generating a germline version of the anti-Ars antibody which had a
two-hundred fold loss in activity. The full affinity of the anti-Ars
antibody could be recovered by restoring only three of the nineteen
somatic mutations, demonstrating that many somatic mutations may be
permitted that do not contribute to antigen binding activity.

[0444] The result can be explained in part by the nature of antibody
diversity itself. Immature B-cells may produce initially low affinity
antibodies that recognize a number of self or non-self antigens.
Moreover, antibodies may undergo in the course of affinity maturation
sequence variations that may cause self-reactivity. Hypermutation of such
low affinity antibodies may serve to abolish self-reactivity ("negative
selection") and increase affinity for the foreign antigen. Therefore, the
analysis of primary and structural data of a large number of antibodies
does not provide a method of predicting either (1) the role of somatic
hyper-mutation sites in the affinity maturation process versus the
process of decreasing affinity towards unwanted antigens, or (2) how a
given amino acid contributes to the properties of a specific
antigen-antibody pair.

[0445] Other attempts to address the role of specific amino acid residues
in antigen recognition were made by analyzing a number of crystal
structures of antigen-antibody complexes (MacCallum et al. (1996) J. Mol.
Biol. 262: 732-745). The potential role of positions located within and
outside the CDRs was indicated. Positions in CDRs involved in antigen
binding in more than 10 of 26 analyzed structures included H31, H33, H50,
H52, H53, H54, H56, H58, H95, H96, H97, H98 and H100 in the heavy chain
and L30A, L32, L91, L92, L93, L94, L96 in the light chain. However, the
authors noted that prediction of antigen contacts using these and other
structural data may over and under predict contact positions, leading to
the speculation that a different strategy may have to be applied to
different antigens.

[0446] Pini et al. describe randomizing multiple residues in antibody CDR
sequences in a large phage display library to rapidly increase antibody
affinity (Pini et al. (1998) J. Biol. Chem. 273: 21769-21776). However,
the high affinity antibodies discussed by Pini et al. had mutations in a
total of eight positions, and a reductionary analysis of which changes
are absolutely required to improve affinity of the antibody becomes
impractical because of the large number of possible combinations to be
tested for the smallest number of amino acids required.

[0447] Furthermore, randomizing multiple residues may not necessarily
preserve other desired properties of the antibody. Desirable properties
or characteristics of an antibody are art-recognized and include for
example, preservation of non-cross reactivity, e.g., with other proteins
or human tissues and preservation of antibody sequences that are close to
human germline immunoglobulin sequences improvement of neutralization
potency. Other desirable properties or characteristics include ability to
preserve species cross reactivity, ability to preserve epitope
specificity and ability to preserve high expression levels of protein in
mammalian cells. The desirable properties or characteristics can be
observed or measured using art-recognized techniques including but not
limited to ELISA, competitive ELISA, in vitro and in vivo neutralization
assays (see e.g. Example 3), immunohistochemistry with tissue sections
from different sources including human, primate or other sources as the
need may be, and studies to expression in mammalian cells using transient
expression or stable expression.

[0448] In addition, the method of Pini et al may introduce more changes
than the minimal number actually required to improve affinity and may
lead to the antibodies triggering anti-human-antibody (HAMA) formation in
human subjects. Further, as discussed elsewhere, the phage display as
demonstrated here, or other related method including ribosome display may
not work appropriately upon reaching certain affinities between antibody
and antigen and the conditions required to reach equilibrium may not be
established in a reasonable time frame because of additional interactions
including interactions with other phage or ribosome components and the
antigen.

[0449] The ordinarily skilled artisan may glean interesting scientific
information on the origin of antibody diversity from the teachings of the
references discussed above. The present invention, however, provides a
method for increasing antibody affinity of a specific antigen-antibody
pair while preserving other relevant features or desirable
characteristics of the antibody. This is especially important when
considering the desirability of imparting a multitude of different
characteristics on a specific antibody including antigen binding.

[0450] If the starting antibody has desirable properties or
characteristics which need to be retained, a selective mutagenesis
approach can be the best strategy for preserving these desirable
properties while improving the activity of the antibody. For example, in
the mutagenesis of Y61, the aim was to increase affinity for hIL-12, and
to improve the neutralization potency of the antibody while preserving
desired properties. Desired properties of Y61 included (1) preservation
of non-cross reactivity with other proteins or human tissues, (2)
preservation of fine epitope specificity, i.e. recognizing a p40 epitope
preferably in the context of the p70 (p40/p35) heterodimer, thereby
preventing binding interference from free soluble p40; and (3) generation
of an antibody with heavy and light chain amino acid sequences that were
as close as possible to their respective germline immunoglobulin
sequences.

[0451] In one embodiment, the method of the invention provides a selective
mutagenesis approach as a strategy for preserving the desirable
properties or characteristics of the antibody while improving the
affinity and/or neutralization potency. The term "selective mutagenesis
approach" is as defined above and includes a method of individually
mutating selected amino acid residues. The amino acid residues to be
mutated may first be selected from preferred selective mutagenesis
positions, then from contact positions, and then from hypermutation
positions. The individual selected position can be mutated to at least
two other amino acid residue and the effect of the mutation both on the
desired properties of the antibody, and improvement in antibody activity
is determined.

[0452] The Selective Mutagenesis approach comprises the steps of:

[0453] selecting candidate positions in the order 1) preferred selective
mutagenesis positions; 2) contact positions; 3) hypermutation positions
and ranking the positions based on the location of the position within
the heavy and light chain variable regions of an antibody (CDR3 preferred
over CDR2 preferred over CDR1);

[0454] individually mutating candidate preferred selective mutagenesis
positions, hypermutation and/or contact positions in the order of
ranking, to all possible other amino acid residues and analyzing the
effect of the individual mutations on the activity of the antibody in
order to determine activity enhancing amino acid residues;

[0455] if necessary, making stepwise combinations of the individual
activity enhancing amino acid residues and analyzing the effect of the
various combinations on the activity of the antibodies; selecting mutant
antibodies with activity enhancing amino acid residues and ranking the
mutant antibodies based on the location and identity of the amino acid
substitutions with regard to their immunogenic potential. Highest ranking
is given to mutant antibodies that comprise an amino acid sequence which
nearly identical to a variable region sequence that is described in a
germline database, or has an amino acid sequence that is comparable to
other human antibodies. Lower ranking is given to mutant antibodies
containing an amino acid substitution that is rarely encountered in
either germline sequences or the sequences of other human antibodies. The
lowest ranking is given to mutant antibodies with an amino acid
substitution that has not been encountered in a germline sequence or the
sequence of another human antibody. As set forth above, mutant antibodies
comprising at least one activity enhancing amino acid residue located in
CDR3 is preferred over CDR2 which is preferred over CDR1. The CDRs of the
heavy chain variable regions are preferred over those of the light chain
variable region.

[0456] The mutant antibodies can also be studied for improvement in
activity, e.g. when compared to their corresponding parental antibody.
The improvement in activity of the mutant antibody can be determined for
example, by neutralization assays, or binding specificity/affinity by
surface plasmon resonance analysis (see Example 3). Preferably, the
improvement in activity can be at least 2-20 fold higher than the
parental antibody. The improvement in activity can be at least "x1"
to "x2" fold higher than the parental antibody wherein "x1" and
"x2" are integers between and including 2 to 20, including ranges
within the state range, e.g. 2-15, e.g. 5-10.

[0457] The mutant antibodies with the activity enhancing amino acid
residue also can be studied to determine whether at least one other
desirable property has been retained after mutation. For example, with
anti-hIL-12 antibodies testing for, (1) preservation of non-cross
reactivity with other proteins or human tissues, (2) preservation of
epitope recognition, i.e. recognizing a p40 epitope preferably in the
context of the p70 (p40/p35) heterodimer, thereby preventing binding
interference from free soluble p40; and (3) generation of antibodies with
heavy and light chain amino acid sequences that were as close as possible
to their respective germline immunoglobulin sequences, and determining
which would be least likely to elicit a human immune response based on
the number of differences from the germline sequence. The same
observations can be made on an antibody having more than one activity
enhancing amino acid residues, e.g. at least two or at least three
activity enhancing amino acid residues, to determine whether retention of
the desirable property or characteristic has occurred.

[0458] An example of the use of a "selective mutagenesis approach", in the
mutagenesis of Y61 is described below. The individual mutations
H31S→E, L50→Y, or L94G→Y each improved
neutralization activity of the antibody. However, when combination clones
were tested, the activity of the combined clone
H31S→E+L50→Y+L94G→Y was no better than
L50→Y+L94G→Y (J695). Therefore, changing the germline amino
acid residue Ser to Glu at position 31 of CDR1 was unnecessary for the
improved activity of J695 over Y61. The selective mutagenesis approach
therefore, identified the minimal number of changes that contributed to
the final activity, thereby reducing the immunogenic potential of the
final antibody and preserving other desired properties of the antibody.

[0459] Isolated DNA encoding the VH and VL produced by the selected
mutagenesis approach can be converted into full length antibody chain
genes, to Fab fragment genes as to a scFV gene, as described in section
IV. For expression of VH and VL regions produced by the selected
mutagenesis approach, expression vectors encoding the heavy and light
chain can be transfected into variety host cells as described in detail
in section IV. Preferred host cells include either prokaryotic host
cells, for example, E. coli, or eukaryotic host cells, for example, yeast
cells, e.g., S. cerevisae. Most preferred eukaryotic host cells are
mammalian host cells, described in detail in section IV.

[0460] The selective mutagenesis approach provides a method of producing
antibodies with improved activities without prior affinity maturation of
the antibody by other means. The selective mutagenesis approach provides
a method of producing antibodies with improved affinities which have been
subject to back mutations. The selective mutagenesis approach also
provides a method of improving the activity of affinity matured
antibodies.

[0461] The skilled artisan will recognize that the selective mutagenesis
approach can be used in standard antibody manipulation techniques known
in the art. Examples include, but are not limited to, CDR grafted
antibodies, chimeric antibodies, scFV fragments, Fab fragments of a full
length antibodies and human antibodies from other sources, e.g.,
transgenic mice.

[0463] In the methods of the invention, antibodies or antigen binding
portions thereof are further modified by altering individual positions in
the CDRs of the HCVR and/or LCVR. Although these modifications can be
made in phage-displayed antibodies, the method is advantageous in that it
can be performed with antibodies that are expressed in other types of
host systems, such as bacterial, yeast or mammalian cell expression
systems. The individual positions within the CDRs selected for
modification are based on the positions being a contact and/or
hypermutation position.

[0466] In general, the method of the invention involves selecting a
particular preferred selective mutagenesis position, contact and/or
hypermutation position within a CDR of the heavy or light chain of a
parent antibody of interest, or antigen binding portion thereof, randomly
mutagenizing that individual position (e.g., by genetic means using a
mutagenic oligonucleotide to generate a "mini-library" of modified
antibodies), or mutating a position to specific desired amino acids, to
identify activity enhancing amino acid residues expressing, and purifying
the modified antibodies (e.g., in a non-phage display host system),
measuring the activity of the modified antibodies for antigen (e.g., by
measuring koff rates by BIAcore analysis), repeating these steps for
other CDR positions, as necessary, and combining individual mutations
shown to have improved activity and testing whether the combination(s)
generate an antibody with even greater activity (e.g., affinity or
neutralizing potency) than the parent antibody, or antigen-binding
portion thereof.

[0467] Accordingly, in one embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0470] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;

[0471] d) evaluating the activity of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof;

[0473] f) combining, in the parent antibody, or antigen-binding portion
thereof, individual mutations shown to have improved activity, to form
combination antibodies, or antigen-binding portions thereof; and

[0474] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained. Preferably,
the selected antibody or antibodies have an improved activity without
loss or with retention of at least one desirable characteristic or
property of the parental antibody as described above. The desirable
characteristic or property can be measured or observed by the ordinarily
skilled artisan using art-recognized techniques.

[0478] b) selecting a preferred selective mutagenesis position, contact or
hypermutation position within a complementarity determining region (CDR)
for mutation;

[0479] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;

[0487] b) selecting a preferred selective mutagenesis position, contact or
hypermutation position within a complementarity determining region (CDR)
for mutation;

[0488] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof;

[0494] Following mutagenesis of individual selected positions, mutated
clones can be sequenced to identify which amino acid residues have been
introduced into the selected position in each clone. A small number of
clones (e.g., about 24) can be selected for sequencing, which
statistically should yield 10-15 unique antibodies, whereas larger
numbers of clones (e.g., greater than 60) can be sequenced to ensure that
antibodies with every possible substitution at the selected position are
identified.

[0495] In one embodiment, contact and/or hypermutation positions within
the CDR3 regions of the heavy and/or light chains are first selected for
mutagenesis. However, for antibodies that have already been affinity
matured in vitro by random mutagenesis of the CDR3 regions via phage
display selection, it may be preferably to first select contact and/or
hypermutation positions within CDR1 or CDR2 of the heavy and/or light
chain.

[0496] In a more preferred embodiment, preferred selective mutagenesis
positions within the CDR3 regions of the heavy and/or light chains are
first selected for mutagenesis. However, for antibodies that have already
been affinity matured in vitro by random mutagenesis of the CDR3 regions
via phage display selection, it may be preferably to first select
preferred selective mutagenesis positions within CDR1 or CDR2 of the
heavy and/or light chain.

[0497] In another preferred embodiment, the optimization of a selected
antibody by the selective mutagenesis approach is done sequentially as
follows: preferred selective mutagenesis positions selected from the
group consisting of H30, H31, H31B, H32, H33, H52, H56, H58, L30, L31,
L32, L50, L91, L92, L93, L94 are mutated first to at least 2 other amino
acids each (preferably 5-14 other amino acids) and the resulting
antibodies are characterized for increased affinity, neutralization
potency (and possibly also for at least one other retained characteristic
or property discussed elsewhere). If a mutation of a single preferred
selective mutagenesis position does not increase the affinity or
neutralization potency at all or sufficiently and if even the combination
of multiple activity enhancing amino acids replacing amino acids in
preferred selective mutagenesis positions does not result in an
combination antibody which meets the target activity (including affinity
and/or neutralization potency), additional amino acid residues will be
selected for selective mutagenesis from the group consisting of H35, H50,
H53, H54, H95, H96, H97, H98, L30A and L96 are mutated to at least 2
other amino acids each (preferably 5-14 other amino acids) and the
resulting antibodies are characterized for increased affinity,
neutralization potency (and possibly also for at least one other retained
characteristic or property discussed elsewhere).

[0498] If a mutation of a single amino acid residue selected from the
group consisting of H35, H50, H53, H54, H95, H96, H97, H98, L30A and L96
does not increase the activity (including affinity and/or neutralization
potency) at all or not sufficiently and if even the combination of
multiple activity enhancing amino acids replacing amino acids in those
positions does not result in an combination antibody which meets the
targeted activity (including affinity and/or target neutralization
potency), additional amino acid residues will be selected for selective
mutagenesis from the group consisting of H33B, H52B, L31A and are mutated
to at least 2 other amino acids each (preferably 5-14 other amino acids)
and the resulting antibodies are characterized for increased affinity,
neutralization potency (and possibly also for at least one other retained
characteristic or property discussed elsewhere).

[0499] It should be understood that the sequential selective mutagenesis
approach may end at any of the steps outline above as soon as an antibody
with the desired activity (including affinity and neutralization potency)
has been identified. If mutagenesis of the preselected positions has
identified activity enhancing amino acids residues but the combination
antibody still do not meet the targets set for activity (including
affinity and neutralization potency) and/or if the identified activity
enhancing amino acids also affect other desired characteristics and are
therefore not acceptable, the remaining CDR residues may be subjected to
mutagenesis (see section IV).

[0500] The method of the invention can be used to improve activity of an
antibody, or antigen binding portion thereof, to reach a predetermined
target activity (e.g. a predetermined affinity and/or neutralization
potency, and/or a desired property or characteristic).

[0501] Accordingly, the invention provides a method of improving the
activity of an antibody, or antigen-binding portion thereof, to attain a
predetermined target activity, comprising:

[0504] c) individually mutating the selected preferred selective
mutagenesis position to at least two other amino acid residues to hereby
create a first panel of mutated antibodies, or antigen binding portions
thereof;

[0505] d) evaluating the activity of the first panel of mutated
antibodies, or antigen binding portions thereof to determined if mutation
of a single selective mutagenesis position produces an antibody or
antigen binding portion thereof with the predetermined target activity or
a partial target activity;

[0506] e) combining in a stepwise fashion, in the parent antibody, or
antigen binding portion thereof, individual mutations shown to have an
improved activity, to form combination antibodies, or antigen binding
portions thereof.

[0507] f) evaluating the activity of the combination antibodies, or
antigen binding portions thereof to determined if the combination
antibodies, or antigen binding portions thereof have the predetermined
target activity or a partial target activity.

[0508] g) if steps d) or f) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity, or
result an antibody with only a partial activity, additional amino acid
residues selected from the group consisting of H35, H50, H53, H54, H95,
H96, H97, H98, L30A and L96 are mutated to at least two other amino acid
residues to thereby create a second panel of mutated antibodies or
antigen-binding portions thereof;

[0509] h) evaluating the activity of the second panel of mutated
antibodies or antigen binding portions thereof, to determined if mutation
of a single amino acid residue selected from the group consisting of H35,
H50, H53, H54, H95, H96, H97, H98, L30A and L96 results an antibody or
antigen binding portion thereof, having the predetermined target activity
or a partial activity;

[0510] i) combining in stepwise fashion in the parent antibody, or
antigen-binding portion thereof, individual mutations of step g) shown to
have an improved activity, to form combination antibodies, or antigen
binding portions thereof;

[0511] j) evaluating the activity of the combination antibodies or antigen
binding portions thereof, to determined if the combination antibodies, or
antigen binding portions thereof have the predetermined target activity
or a partial target activity;

[0512] k) if steps h) or j) do not result in an antibody or antigen
binding portion thereof having the predetermined target activity, or
result in an antibody with only a partial activity, additional amino acid
residues selected from the group consisting of H33B, H52B and L31A are
mutated to at least two other amino acid residues to thereby create a
third panel of mutated antibodies or antigen binding portions thereof;

[0513] l) evaluating the activity of the third panel of mutated antibodies
or antigen binding portions thereof, to determine if a mutation of a
single amino acid residue selected from the group consisting of H33B,
H52B and L31A resulted in an antibody or antigen binding portion thereof,
having the predetermined target activity or a partial activity;

[0514] m) combining in a stepwise fashion in the parent antibody, or
antigen binding portion thereof, individual mutation of step k) shown to
have an improved activity, to form combination antibodies, or antigen
binding portions, thereof,

[0515] n) evaluating the activity of the combination antibodies or
antigen-binding portions thereof, to determine if the combination
antibodies, or antigen binding portions thereof have the predetermined
target activity to thereby produce an antibody or antigen binding portion
thereof with a predetermined target activity.

[0516] A number of mutagenesis methods can be used, including PCR
assembly, Kunkel (dut-ung-) and thiophosphate (Amersham Sculptor kit)
oligonucleotide-directed mutagenesis.

[0517] A wide variety of host expression systems can be used to express
the mutated antibodies, including bacterial, yeast, baculoviral and
mammalian expression systems (as well as phage display expression
systems). An example of a suitable bacterial expression vector is
pUC119(Sfi). Other antibody expression systems are known in the art
and/or are described below in section IV.

[0518] The modified antibodies, or antigen binding portions thereof,
produced by the method of the invention can be identified without the
reliance on phage display methods for selection. Accordingly, the method
of the invention is particularly advantageous for improving the activity
of a recombinant parent antibody or antigen-binding portion thereof, that
was obtained by selection in a phage-display system but whose activity
cannot be further improved by mutagenesis in the phage-display system.

[0519] Accordingly, in another embodiment, the invention provides a method
for improving the affinity of an antibody, or antigen-binding portion
thereof, comprising:

[0520] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0522] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0523] d) evaluating the activity of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof;

[0525] f) combining, in the parent antibody, or antigen-binding portion
thereof, individual mutations shown to have improved activity, to form
combination antibodies, or antigen-binding portions thereof; and

[0526] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0528] With available methods it is not possible or it is extremely
laborious to derive an antibody with increased binding affinity and
neutralization potency while retaining other properties or
characteristics of the antibodies as discussed above. The method of this
invention, however, can readily identify such antibodies. The antibodies
subjected to the method of this invention can come from any source.

[0529] Therefore, in another embodiment, the invention provides a method
for improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0532] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expressing said panel
in an appropriate expression system;

[0534] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristics,
wherein the property or characteristic is one that needs to be retained
in the antibody; until an antibody, or antigen-binding portion thereof,
with an improved activity and at least one retained property or
characteristic, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.

[0536] In another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58,
L30, L31, L32, L53 and L93 and the other characteristic is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0537] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of non-crossreactivity
with other proteins or human tissues, 2) preservation of epitope
recognition, i.e. recognizing p40 epitope preferably in the context of
the p70 p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence.

[0538] In a more preferred embodiment, the contact positions are selected
from the group consisting of L50 and L94 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence.

[0539] If therefore, the affinity of an antibody for a specific antigen
should be improved, but where the phage display (or related system
including ribosome display) method is no longer applicable, and other
desirable properties or characteristics should be retained, the method of
the invention can be used. Accordingly, in another embodiment, the
invention provides a method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:

[0540] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0542] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0544] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristic,
wherein the property or characteristic is one that needs to be retained,
until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0546] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and at least one retained property or
characteristic, to form combination antibodies, or antigen-binding
portions thereof; and

[0547] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one retained
other property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0549] In another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58,
L30, L31, L32, L53 and L93 and the other characteristic is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0550] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of non-crossreactivity
with other proteins or human tissues, 2) preservation of epitope
recognition, i.e. recognizing p40 epitope preferably in the context of
the p70 p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence.

[0551] In a more preferred embodiment, the contact positions are selected
from the group consisting of L50 and L94 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence.

[0552] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0553] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0555] c) individually mutating said selected preferred selective
mutagenesis position, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0557] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristic,
wherein the property or characteristic is one that needs to be retained,
until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained property or characteristic, relative
to the parent antibody, or antigen-binding portion thereof, is obtained.

[0559] In another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58,
L30, L31, L32, L53 and L93 and the other characteristic is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0560] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of non-crossreactivity
with other proteins or human tissues, 2) preservation of epitope
recognition, i.e. recognizing p40 epitope preferably in the context of
the p70 p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence.

[0561] In a more preferred embodiment, the contact positions are selected
from the group consisting of L50 and L94 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence.

[0562] In another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0563] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0565] c) individually mutating said selected preferred selective
mutagenesis positions, contact or hypermutation position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof, and expressing said
panel in a non-phage display system;

[0567] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof for at least one other property or characteristic,
wherein the property or characteristic is one that needs to be retained,
until an antibody, or antigen-binding portion thereof, with an improved
activity and at least one retained characteristic, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0569] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and at least on retained other
characteristic, to form combination antibodies, or antigen-binding
portions thereof; and

[0570] h) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity and at least one retained
property or characteristic, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0572] In another preferred embodiment, the hypermutation positions are
selected from the group consisting of H30, H31, H31B, H32, H52, H56, H58,
L30, L31, L32, L53 and L93 and the other characteristic is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0573] In a more preferred embodiment the residues for selective
mutagenesis are selected from the preferred selective mutagenesis
positions from the group consisting of H30, H31, H31B, H32, H33, H52,
H56, H58, L30, L31, L32, L50, L91, L92, L93, L94 and the other
characteristic is selected from 1) preservation of non-crossreactivity
with other proteins or human tissues, 2) preservation of epitope
recognition, i.e. recognizing p40 epitope preferably in the context of
the p70 p40/p35 heterodimer preventing binding interference from free,
soluble p40 and/or 3) to produce an antibody with a close to germline
immunoglobulin sequence.

[0574] In a more preferred embodiment, the contact positions are selected
from the group consisting of L50 and L94 and the other characteristic is
selected from 1) preservation of non-crossreactivity with other proteins
or human tissues, 2) preservation of epitope recognition, i.e.
recognizing p40 epitope preferably in the context of the p70 p40/p35
heterodimer preventing binding interference from free, soluble p40 and/or
3) to produce an antibody with a close to germline immunoglobulin
sequence.

IV. Modifications of Other CDR Residues

[0575] Ultimately, all CDR residues in a given antibody-antigen pair
identified by any means to be required as activity enhancing amino acid
residues and/or required directly or indirectly for binding to the
antigen and/or for retaining other desirable properties or
characteristics of the antibody. Such CDR residues are referred to as
"preferred selective mutagenesis positions". It should be noted that in
specific circumstances that preferred selective mutagenesis residues can
be identified also by other means including co-crystallization of
antibody and antigen and molecular modeling.

[0576] If the preferred attempts to identify activity enhancing amino
acids focussing on the preferred selective mutagenesis positions, contact
or hypermutation positions described above are exhausted, or if
additional improvements are required, the remaining CDR residues may be
modified as described below. It should be understood that the antibody
could already be modified in any one or more contact or hypermutation
positions according to the embodiments discussed above but may require
further improvements. Therefore, in another embodiment, the invention
provides a method for improving the activity of an antibody, or
antigen-binding portion thereof, comprising:

[0579] c) individually mutating said selected position e.g., to at least
two other amino acid residues to thereby create a mutated antibody or a
panel of mutated antibodies, or antigen-binding portions thereof;

[0580] d) evaluating the activity of the mutated antibody or the panel of
mutated antibodies, or antigen-binding portions thereof, relative to the
parent antibody or antigen-binding portion thereof thereby identifying an
activity enhancing amino acid residue;

[0581] e) evaluating the mutated antibody or the panel of mutated
antibodies, or antigen-binding portions thereof, relative to the parent
antibody or antigen-binding portion thereof, for changes in at least one
other property or characteristic until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0582] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0583] If mutagenesis of a single residue is not sufficient other residues
can be included; therefore, in another embodiment, the invention provides
a method for improving the activity of an antibody, or antigen-binding
portion thereof, comprising:

[0589] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity, to form combination antibodies, or
antigen-binding portions thereof; and

[0590] g) evaluating the activity of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.

[0591] If the preferred attempts to identify activity enhancing amino
acids focussing on the contact or hypermutation positions described above
are exhausted, or if additional improvements are required, and the
antibody in question can not further be optimized by mutagenesis and
phage display (or related ribosome display) methods the remaining CDR
residues may be modified as described below. It should be understood that
the antibody could already be modified in any one or more preferred
selective mutagenesis position, contact or hypermutation positions
according to the embodiments discussed above but may require further
improvements.

[0592] Therefore, in another embodiment, the invention provides a method
for improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0593] a) providing a recombinant parent antibody or antigen-binding
portion thereof; that was obtained by selection in a phage-display system
but whose activity cannot be further improved by mutagenesis in said
phage-display system;

[0595] c) individually mutating said selected contact or hypermutation
position to at least two other amino acid residues to thereby create a
panel of mutated antibodies, or antigen-binding portions thereof, and
expressing said panel in a non-phage display system;

[0597] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic, until an antibody, or antigen-binding portion thereof,
with an improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0598] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence.

[0599] If a single mutagenesis is not sufficient to increase the affinity
of the antibody other residues may be included in the mutagenesis.
Therefore, in another embodiment, the invention provides a method for
improving the activity of an antibody, or antigen-binding portion
thereof, comprising:

[0600] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0602] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0605] g) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity, to form combination antibodies, or
antigen-binding portions thereof; and

[0606] h) evaluating the activity and other property or characteristic of
the combination antibodies, or antigen-binding portions thereof with two
activity enhancing amino acid residues, relative to the parent antibody
or antigen-binding portion thereof; until an antibody, or antigen-binding
portion thereof, with an improved activity, relative to the parent
antibody, or antigen-binding portion thereof, is obtained.

[0607] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0608] The preferred attempts to identify activity enhancing amino acids
focussing on the preferred selective mutagenesis positions, contact or
hypermutation positions described may be exhausted, or additional
improvements may be required, and it is important to retain other
properties or characteristics of the antibody.

[0609] Therefore, in another embodiment, the invention provides a method
for improving the activity of an antibody, or antigen-binding portion
thereof, without affecting other characteristics, comprising:

[0614] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic until an antibody, or antigen-binding portion thereof,
with an improved activity and retained other property or characteristic,
relative to the parent antibody, or antigen-binding portion thereof, is
obtained.

[0615] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0616] If mutagenesis of a single residue is not sufficient other residues
can be included; therefore, in another embodiment, the invention provides
a method for improving the activity of an antibody, or antigen-binding
portion thereof, comprising:

[0621] e) evaluating the panel of mutated antibodies or antigen-binding
portions thereof, relative to the parent antibody or antigen-portion
thereof, for changes in at least one other characteristic or property;

[0623] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and not affecting at least one other
property or characteristic, to form combination antibodies, or
antigen-binding portions thereof, and

[0624] g) evaluating the activity and the retention of at least one other
property or characteristic of the combination antibodies, or
antigen-binding portions thereof with two activity enhancing amino acid
residues, relative to the parent antibody or antigen-binding portion
thereof until an antibody, or antigen-binding portion thereof, with an
improved activity and at least one retained other property or
characteristic, relative to the parent antibody, or antigen-binding
portion thereof, is obtained.

[0625] Mutagenesis of the preferred selective mutagenesis position,
contact and hypermutation residues may not have increased the affinity of
the antibody sufficiently, and mutagenesis and the phage display method
(or related ribosome display method) may no longer be useful and at least
one other characteristic or property of the antibody should be retained.

[0626] Therefore, in another embodiment the invention provides a method to
improve the affinity of an antibody or antigen-binding portion thereof,
comprising:

[0627] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0629] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0631] e) evaluating the panel of mutated antibodies, or antigen-binding
portions thereof, relative to the parent antibody or antigen-binding
portion thereof, for changes in at least one other property or
characteristic until an antibody, or antigen-binding portion thereof,
with an improved activity, relative to the parent antibody, or
antigen-binding portion thereof, is obtained.

[0632] Preferably, the other characteristic or property is selected from
1) preservation of non-crossreactivity with other proteins or human
tissues, 2) preservation of epitope recognition, i.e. recognizing p40
epitope preferably in the context of the p70 p40/p35 heterodimer
preventing binding interference from free, soluble p40 and/or 3) to
produce an antibody with a close to germline immunoglobulin sequence

[0633] If mutagenesis of a single residue is not sufficient other residues
can be included; therefore, in another embodiment, the invention provides
a method for improving the activity of an antibody, or antigen-binding
portion thereof, comprising:

[0634] a) providing a parent antibody or antigen-binding portion thereof
that was obtained by selection in a phage-display system but whose
activity cannot be further improved by mutagenesis in said phage-display
system;

[0636] c) individually mutating said selected position to at least two
other amino acid residues to thereby create a panel of mutated
antibodies, or antigen-binding portions thereof and expression in a
non-phage display system;

[0637] d) evaluating the activity and retention of at least one other
property or characteristic of the panel of mutated antibodies, or
antigen-binding portions thereof, relative to the parent antibody or
antigen-binding portion thereof, thereby identifying an activity
enhancing amino acid residue;

[0639] f) combining, in the parent antibody, or antigen-binding portion
thereof, at least two individual activity enhancing amino acid residues
shown to have improved activity and not to affect at least one other
property or characteristic, to form combination antibodies, or
antigen-binding portions thereof, and

[0640] g) evaluating the activity and retention of at least one property
or characteristic of the combination antibodies, or antigen-binding
portions thereof with two activity enhancing amino acid residues,
relative to the parent antibody or antigen-binding portion thereof until
an antibody, or antigen-binding portion thereof, with an improved
activity and at least one other retained characteristic or property,
relative to the parent antibody, or antigen-binding portion thereof, is
obtained.

V. Expression of Antibodies

[0641] An antibody, or antibody portion, of the invention can be prepared
by recombinant expression of immunoglobulin light and heavy chain genes
in a host cell. To express an antibody recombinantly, a host cell is
transfected with one or more recombinant expression vectors carrying DNA
fragments encoding the immunoglobulin light and heavy chains of the
antibody such that the light and heavy chains are expressed in the host
cell and, preferably, secreted into the medium in which the host cells
are cultured, from which medium the antibodies can be recovered. Standard
recombinant DNA methodologies are used to obtain antibody heavy and light
chain genes, incorporate these genes into recombinant expression vectors
and introduce the vectors into host cells, such as those described in
Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory
Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M.
et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing
Associates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

[0642] To obtain a DNA fragment encoding the heavy chain variable region
of Joe 9 wt or a Joe 9 wt-related antibody, antibodies specific for human
IL-12 were screened from human libraries and mutated, as described in
section II. Once DNA fragments encoding Joe 9 wt or Joe 9 wt-related VH
and VL segments are obtained, mutagenesis of these sequences is carried
out by standard methods, such as PCR site directed mutagenesis
(PCR-mediated mutagenesis in which the mutated nucleotides are
incorporated into the PCR primers such that the PCR product contains the
mutations) or other site-directed mutagenesis methods. Human IL-12
antibodies that displayed a level of activity and binding
specificity/affinity that was desirable, for example J695, were further
manipulated by standard recombinant DNA techniques, for example to
convert the variable region genes to full-length antibody chain genes, to
Fab fragment genes or to a scFv gene. In these manipulations, a VL- or
VH-encoding DNA fragment is operatively linked to another DNA fragment
encoding another protein, such as an antibody constant region or a
flexible linker. The term "operatively linked", as used in this context,
is intended to mean that the two DNA fragments are joined such that the
amino acid sequences encoded by the two DNA fragments remain in-frame.

[0643] The isolated DNA encoding the VH region can be converted to a
full-length heavy chain gene by operatively linking the VH-encoding DNA
to another DNA molecule encoding heavy chain constant regions (CH1, CH2
and CH3). The sequences of human heavy chain constant region genes are
known in the art (see e.g., Kabat, E. A., et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department of
Health and Human Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR amplification.
The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA,
IgE, IgM or IgD constant region and any allotypic variant therein as
described in Kabat (Kabat, E. A., et al. (1991) Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and
Human Services, NIH Publication No. 91-3242), but most preferably is an
IgG1 or IgG4 constant region. For a Fab fragment heavy chain gene, the
VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region.

[0644] The isolated DNA encoding the VL region can be converted to a
full-length light chain gene (as well as a Fab light chain gene) by
operatively linking the VL-encoding DNA to another DNA molecule encoding
the light chain constant region, CL. The sequences of human light chain
constant region genes are known in the art (see e.g., Kabat, E. A., et
al. (1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH Publication
No. 91-3242) and DNA fragments encompassing these regions can be obtained
by standard PCR amplification. The light chain constant region can be a
kappa or lambda constant region, but most preferably is a lambda constant
region.

[0646] To express the antibodies, or antibody portions of the invention,
DNAs encoding partial or full-length light and heavy chains, obtained as
described above, are inserted into expression vectors such that the genes
are operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is intended to
mean that an antibody gene is ligated into a vector such that
transcriptional and translational control sequences within the vector
serve their intended function of regulating the transcription and
translation of the antibody gene. The expression vector and expression
control sequences are chosen to be compatible with the expression host
cell used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both genes
are inserted into the same expression vector. The antibody genes are
inserted into the expression vector by standard methods (e.g., ligation
of complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present). Prior
to insertion of the J695 or J695-related light or heavy chain sequences,
the expression vector may already carry antibody constant region
sequences. For example, one approach to converting the J695 or
J695-related VH and VL sequences to full-length antibody genes is to
insert them into expression vectors already encoding heavy chain constant
and light chain constant regions, respectively, such that the VH segment
is operatively linked to the CH segment(s) within the vector and the VL
segment is operatively linked to the CL segment within the vector.
Additionally or alternatively, the recombinant expression vector can
encode a signal peptide that facilitates secretion of the antibody chain
from a host cell. The antibody chain gene can be cloned into the vector
such that the signal peptide is linked in-frame to the amino terminus of
the antibody chain gene. The signal peptide can be an immunoglobulin
signal peptide or a heterologous signal peptide (i.e., a signal peptide
from a non-immunoglobulin protein).

[0647] In addition to the antibody chain genes, the recombinant expression
vectors of the invention carry regulatory sequences that control the
expression of the antibody chain genes in a host cell. The term
"regulatory sequence" is intended to include promoters, enhancers and
other expression control elements (e.g. polyadenylation signals) that
control the transcription or translation of the antibody chain genes.
Such regulatory sequences are described, for example, in Goeddel; Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San
Diego, Calif. (1990). It will be appreciated by those skilled in the art
that the design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of the host
cell to be transformed, the level of expression of protein desired, etc.
Preferred regulatory sequences for mammalian host cell expression include
viral elements that direct high levels of protein expression in mammalian
cells, such as promoters and/or enhancers derived from cytomegalovirus
(CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such
as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major
late promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.
Pat. No. 4,968,615 by Schaffner et al., U.S. Pat. No. 5,464,758 by Bujard
et al. and U.S. Pat. No. 5,654,168 by Bujard et al.

[0648] In addition to the antibody chain genes and regulatory sequences,
the recombinant expression vectors of the invention may carry additional
sequences, such as sequences that regulate replication of the vector in
host cells (e.g., origins of replication) and selectable marker genes.
The selectable marker gene facilitates selection of host cells into which
the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216,
4,634,665 and 5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector has been
introduced. Preferred selectable marker genes include the dihydrofolate
reductase (DHFR) gene (for use in dhfr- host cells with methotrexate
selection/amplification) and the neo gene (for G418 selection).

[0649] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a host
cell by standard techniques. The various forms of the term "transfection"
are intended to encompass a wide variety of techniques commonly used for
the introduction of exogenous DNA into a prokaryotic or eukaryotic host
cell, e.g., electroporation, calcium-phosphate precipitation,
DEAE-dextran transfection and the like. Although it is theoretically
possible to express the antibodies of the invention in either prokaryotic
or eukaryotic host cells, expression of antibodies in eukaryotic cells,
and most preferably mammalian host cells, is the most preferred because
such eukaryotic cells, and in particular mammalian cells, are more likely
than prokaryotic cells to assemble and secrete a properly folded and
immunologically active antibody. Preferred mammalian host cells for
expressing the recombinant antibodies of the invention include Chinese
Hamster Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a
DHFR selectable marker, e.g., as described in R. J. Kaufman and P. A.
Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and
SP2 cells. When recombinant expression vectors encoding antibody genes
are introduced into mammalian host cells, the antibodies are produced by
culturing the host cells for a period of time sufficient to allow for
expression of the antibody in the host cells or, more preferably,
secretion of the antibody into the culture medium in which the host cells
are grown. Antibodies can be recovered from the culture medium using
standard protein purification methods.

[0650] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the scope of
the present invention. For example, it may be desirable to transfect a
host cell with DNA encoding either the light chain or the heavy chain
(but not both) of an antibody of this invention. Recombinant DNA
technology may also be used to remove some or all of the DNA encoding
either or both of the light and heavy chains that is not necessary for
binding to hIL-12 The molecules expressed from such truncated DNA
molecules are also encompassed by the antibodies of the invention. In
addition, bifunctional antibodies may be produced in which one heavy and
one light chain are an antibody of the invention and the other heavy and
light chain are specific for an antigen other than hIL-12 by crosslinking
an antibody of the invention to a second antibody by standard chemical
crosslinking methods.

[0651] In a preferred system for recombinant expression of an antibody, or
antigen-binding portion thereof, of the invention, a recombinant
expression vector encoding both the antibody heavy chain and the antibody
light chain is introduced into dhfr-CHO cells by calcium
phosphate-mediated transfection. Within the recombinant expression
vector, the antibody heavy and light chain genes are each operatively
linked to enhancer/promoter regulatory elements (e.g., derived from SV40,
CMV, adenovirus and the like, such as a CMV enhancer/AdMLP promoter
regulatory element or an SV40 enhancer/AdMLP promoter regulatory element)
to drive high levels of transcription of the genes. The recombinant
expression vector also carries a DHFR gene, which allows for selection of
CHO cells that have been transfected with the vector using methotrexate
selection/amplification. The selected transformant host cells are culture
to allow for expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular biology
techniques are used to prepare the recombinant expression vector,
transfect the host cells, select for transformants, culture the host
cells and recover the antibody from the culture medium. Antibodies or
antigen-binding portions thereof of the invention can be expressed in an
animal (e.g., a mouse) that is transgenic for human immunoglobulin genes
(see e.g., Taylor, L. D. et al. (1992) Nucl. Acids Res. 20: 6287-6295).
Plant cells can also be modified to create transgenic plants that express
the antibody or antigen binding portion thereof, of the invention.

[0652] In view of the foregoing, another aspect of the invention pertains
to nucleic acid, vector and host cell compositions that can be used for
recombinant expression of the antibodies and antibody portions of the
invention. Preferably, the invention features isolated nucleic acids that
encode CDRs of J695, or the full heavy and/or light chain variable region
of J695. Accordingly, in one embodiment, the invention features an
isolated nucleic acid encoding an antibody heavy chain variable region
that encodes the J695 heavy chain CDR3 comprising the amino acid sequence
of SEQ ID NO: 25. Preferably, the nucleic acid encoding the antibody
heavy chain variable region further encodes a J695 heavy chain CDR2 which
comprises the amino acid sequence of SEQ ID NO: 27. More preferably, the
nucleic acid encoding the antibody heavy chain variable region further
encodes a J695 heavy chain CDR1 which comprises the amino acid sequence
of SEQ ID NO: 29. Even more preferably, the isolated nucleic acid encodes
an antibody heavy chain variable region comprising the amino acid
sequence of SEQ ID NO: 31 (the full VH region of J695).

[0654] The invention also provides recombinant expression vectors encoding
both an antibody heavy chain and an antibody light chain. For example, in
one embodiment, the invention provides a recombinant expression vector
encoding: [0655] a) an antibody heavy chain having a variable region
comprising the amino acid sequence of SEQ ID NO: 31; and [0656] b) an
antibody light chain having a variable region comprising the amino acid
sequence of SEQ ID NO: 32.

[0657] The invention also provides host cells into which one or more of
the recombinant expression vectors of the invention have been introduced.
Preferably, the host cell is a mammalian host cell, more preferably the
host cell is a CHO cell, an NS0 cell or a COS cell. Still further the
invention provides a method of synthesizing a recombinant human antibody
of the invention by culturing a host cell of the invention in a suitable
culture medium until a recombinant human antibody of the invention is
synthesized. The method can further comprise isolating the recombinant
human antibody from the culture medium.

VI. Pharmaceutical Compositions and Pharmaceutical Administration

[0658] The antibodies and antibody-portions of the invention can be
incorporated into pharmaceutical compositions suitable for administration
to a subject. Typically, the pharmaceutical composition comprises an
antibody or antibody portion of the invention and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings, antibacterial
and antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well as
combinations thereof. In many cases, it will be preferable to include
isotonic agents, for example, sugars, polyalcohols such as mannitol,
sorbitol, or sodium chloride in the composition. Pharmaceutically
acceptable carriers may further comprise minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the antibody or
antibody portion.

[0659] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for parenteral
administration. Preferably, the antibody or antibody-portions will be
prepared as an injectable solution containing 0.1-250 mg/ml antibody. The
injectable solution can be composed of either a liquid or lyophilized
dosage form in a flint or amber vial, ampule or pre-filled syringe. The
buffer can be L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not limited
to, sodium succinate, sodium citrate, sodium phosphate or potassium
phosphate. Sodium chloride can be used to modify the toxicity of the
solution at a concentration of 0-300 mM (optimally 150 mM for a liquid
dosage form). Cryoprotectants can be included for a lyophilized dosage
form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable
cryoprotectants include trenhalose and lactose. Bulking agents can be
included for a lyophilized dosage form, principally 1-10% mannitol
(optimally 2-4%). Stabilizers can be used in both liquid and lyophilized
dosage forms, principally 1-50 mM L-Methionine (optimally 5-10 mM). Other
suitable bulking agents include glycine, arginine, can be included as
0-0.05% polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ surfactants.

[0660] In a preferred embodiment, the pharmaceutical composition includes
the antibody at a dosage of about 0.01 mg/kg-10 mg/kg. More preferred
dosages of the antibody include 1 mg/kg administered every other week, or
0.3 mg/kg administered weekly.

[0661] The compositions of this invention may be in a variety of forms.
These include, for example, liquid, semi-solid and solid dosage forms,
such as liquid solutions (e.g., injectable and infusible solutions),
dispersions or suspensions, tablets, pills, powders, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions, such
as compositions similar to those used for passive immunization of humans
with other antibodies. The preferred mode of administration is parenteral
(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a
preferred embodiment, the antibody is administered by intravenous
infusion or injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.

[0662] Therapeutic compositions typically must be sterile and stable under
the conditions of manufacture and storage. The composition can be
formulated as a solution, microemulsion, dispersion, liposome, or other
ordered structure suitable to high drug concentration. Sterile injectable
solutions can be prepared by incorporating the active compound (i.e.,
antibody or antibody portion) in the required amount in an appropriate
solvent with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile vehicle that
contains a basic dispersion medium and the required other ingredients
from those enumerated above. In the case of sterile, lyophilized powders
for the preparation of sterile injectable solutions, the preferred
methods of preparation are vacuum drying and spray-drying that yields a
powder of the active ingredient plus any additional desired ingredient
from a previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size in the
case of dispersion and by the use of surfactants. Prolonged absorption of
injectable compositions can be brought about by including in the
composition an agent that delays absorption, for example, monostearate
salts and gelatin.

[0663] The antibodies and antibody-portions of the present invention can
be administered by a variety of methods known in the art, although for
many therapeutic applications, the preferred route/mode of administration
is subcutaneous injection, intravenous injection or infusion. As will be
appreciated by the skilled artisan, the route and/or mode of
administration will vary depending upon the desired results. In certain
embodiments, the active compound may be prepared with a carrier that will
protect the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible polymers
can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic
acid, collagen, polyorthoesters, and polylactic acid. Many methods for
the preparation of such formulations are patented or generally known to
those skilled in the art. See, e.g., Sustained and Controlled Release
Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978.

[0664] In certain embodiments, an antibody or antibody portion of the
invention may be orally administered, for example, with an inert diluent
or an assimilable edible carrier. The compound (and other ingredients, if
desired) may also be enclosed in a hard or soft shell gelatin capsule,
compressed into tablets, or incorporated directly into the subject's
diet. For oral therapeutic administration, the compounds may be
incorporated with excipients and used in the form of ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound with,
or co-administer the compound with, a material to prevent its
inactivation.

[0665] Supplementary active compounds can also be incorporated into the
compositions. In certain embodiments, an antibody or antibody portion of
the invention is coformulated with and/or coadministered with one or more
additional therapeutic agents that are useful for treating disorders in
which IL-12 activity is detrimental. For example, an anti-hIL-12 antibody
or antibody portion of the invention may be coformulated and/or
coadministered with one or more additional antibodies that bind other
targets (e.g., antibodies that bind other cytokines or that bind cell
surface molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing therapeutic
agents. Such combination therapies may advantageously utilize lower
dosages of the administered therapeutic agents, thus avoiding possible
toxicities or complications associated with the various monotherapies. It
will be appreciated by the skilled practitioner that when the antibodies
of the invention are used as part of a combination therapy, a lower
dosage of antibody may be desirable than when the antibody alone is
administered to a subject (e.g., a synergistic therapeutic effect may be
achieved through the use of combination therapy which, in turn, permits
use of a lower dose of the antibody to achieve the desired therapeutic
effect).

[0667] Preferably, the antibodies of the invention or antigen-binding
portions thereof, are used to treat rheumatoid arthritis, Crohn's
disease, multiple sclerosis, insulin dependent diabetes mellitus and
psoriasis, as described in more detail in section VII.

[0668] A human antibody, or antibody portion, of the invention also can be
administered with one or more additional therapeutic agents useful in the
treatment of autoimmune and inflammatory diseases.

[0669] Antibodies of the invention, or antigen binding portions thereof
can be used alone or in combination to treat such diseases. It should be
understood that the antibodies of the invention or antigen binding
portion thereof can be used alone or in combination with an additional
agent, e.g., a therapeutic agent, said additional agent being selected by
the skilled artisan for its intended purpose. For example, the additional
agent can be a therapeutic agent art-recognized as being useful to treat
the disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent which imparts a
beneficial attribute to the therapeutic composition e.g., an agent which
effects the viscosity of the composition.

[0670] It should further be understood that the combinations which are to
be included within this invention are those combinations useful for their
intended purpose. The agents set forth below are illustrative for
purposes and not intended to be limited. The combinations which are part
of this invention can be the antibodies of the present invention and at
least one additional agent selected from the lists below. The combination
can also include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed composition
can perform its intended function.

[0671] Preferred combinations are non-steroidal anti-inflammatory drug(s)
also referred to as NSAIDS which include drugs like ibuprofen. Other
preferred combinations are corticosteroids including prednisolone; the
well known side-effects of steroid use can be reduced or even eliminated
by tapering the steroid dose required when treating patients in
combination with the anti-IL-12 antibodies of this invention.
Non-limiting examples of therapeutic agents for rheumatoid arthritis with
which an antibody, or antibody portion, of the invention can be combined
include the following: cytokine suppressive anti-inflammatory drug(s)
(CSAIDs); antibodies to or antagonists of other human cytokines or growth
factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15,
IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodies of the
invention, or antigen binding portions thereof, can be combined with
antibodies to cell surface molecules such as CD2, CD3, CD4, CD8, CD25,
CD28, CD30, CD40, CD45, CD69, CD80 (B7.1), CD86 (B7.2), CD90, or their
ligands including CD154 (gp39 or CD40L).

[0672] Preferred combinations of therapeutic agents may interfere at
different points in the autoimmune and subsequent inflammatory cascade;
preferred examples include TNF antagonists like chimeric, humanized or
human TNF antibodies, D2E7, (U.S. application Ser. No. 08/599,226 filed
Feb. 9, 1996), cA2 (Remicade®), CDP 571, anti-TNF antibody fragments
(e.g., CDP870), and soluble p55 or p75 TNF receptors, derivatives
thereof, (p75TNFR1gG (Enbrel®) or p55TNFR1gG (Lenercept), soluble
IL-13 receptor (sIL-13), and also TNFα converting enzyme (TACE)
inhibitors; similarly IL-1 inhibitors (e.g., Interleukin-1-converting
enzyme inhibitors, such as Vx740, or IL-1RA etc.) may be effective for
the same reason. Other preferred combinations include Interleukin 11,
anti-P7s and p-selectin glycoprotein ligand (PSGL). Yet another preferred
combination are other key players of the autoimmune response which may
act parallel to, dependent on or in concert with IL-12 function;
especially preferred are IL-18 antagonists including IL-18 antibodies or
soluble IL-18 receptors, or IL-18 binding proteins. It has been shown
that IL-12 and IL-18 have overlapping but distinct functions and a
combination of antagonists to both may be most effective. Yet another
preferred combination are non-depleting anti-CD4 inhibitors. Yet other
preferred combinations include antagonists of the co-stimulatory pathway
CD80 (B7.1) or CD86 (B7.2) including antibodies, soluble receptors or
antagonistic ligands.

[0675] Preferred examples of therapeutic agents for Crohn's disease in
which an antibody or an antigen binding portion can be combined include
the following: TNF antagonists, for example, anti-TNF antibodies, D2E7
(U.S. application Ser. No. 08/599,226, filed Feb. 9, 1996), cA2
(Remicade®), CDP 571, anti-TNF antibody fragments (e.g., CDP870),
TNFR-Ig constructs (p75TNFRIgG (Enbrel®) and p55TNFRIgG (Lenercept)),
anti-P7s, p-selectin glycoprotein ligand (PSGL), soluble IL-13 receptor
(sIL-13), and PDE4 inhibitors. Antibodies of the invention or antigen
binding portions thereof, can be combined with corticosteroids, for
example, budenoside and dexamethasone. Antibodies of the invention or
antigen binding portions thereof, may also be combined with agents such
as sulfasalazine, 5-aminosalicylic acid and olsalazine, and agents which
interfere with synthesis or action of proinflammatory cytokines such as
IL-1, for example, IL-1β converting enzyme inhibitors (e.g., Vx740)
and IL-Ira. Antibodies of the invention or antigen binding portion
thereof may also be used with T cell signaling inhibitors, for example,
tyrosine kinase inhibitors 6-mercaptopurines. Antibodies of the invention
or antigen binding portions thereof, can be combined with IL-11.

[0677] Preferred examples of therapeutic agents for multiple sclerosis in
which the antibody or antigen binding portion thereof can be combined to
include interferon-0, for example, IFNβ1a and IFNβ1b; copaxone,
corticosteroids, IL-1 inhibitors, TNF inhibitors, and antibodies to CD40
ligand and CD80.

[0678] The pharmaceutical compositions of the invention may include a
"therapeutically effective amount" or a "prophylactically effective
amount" of an antibody or antibody portion of the invention. A
"therapeutically effective amount" refers to an amount effective, at
dosages and for periods of time necessary, to achieve the desired
therapeutic result. A therapeutically effective amount of the antibody or
antibody portion may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the antibody
or antibody portion to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the antibody or antibody portion are outweighed by
the therapeutically beneficial effects. A "prophylactically effective
amount" refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than the
therapeutically effective amount.

[0679] Dosage regimens may be adjusted to provide the optimum desired
response (e.g., a therapeutic or prophylactic response). For example, a
single bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic situation. It
is especially advantageous to formulate parenteral compositions in dosage
unit form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited as
unitary dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms of
the invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular therapeutic or
prophylactic effect to be achieved, and (b) the limitations inherent in
the art of compounding such an active compound for the treatment of
sensitivity in individuals.

[0680] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody portion of
the invention is 0.01-20 mg/kg, more preferably 1-10 mg/kg, even more
preferably 0.3-1 mg/kg. It is to be noted that dosage values may vary
with the type and severity of the condition to be alleviated. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual need
and the professional judgment of the person administering or supervising
the administration of the compositions, and that dosage ranges set forth
herein are exemplary only and are not intended to limit the scope or
practice of the claimed composition.

VII. Uses of the Antibodies of the Invention

[0681] Given their ability to bind to hIL-12, the anti-hIL-12 antibodies,
or portions thereof, of the invention can be used to detect hIL-12 (e.g.,
in a biological sample, such as serum or plasma), using a conventional
immunoassay, such as an enzyme linked immunosorbent assays (ELISA), an
radioimmunoassay (RIA) or tissue immunohistochemistry. The invention
provides a method for detecting hIL-12 in a biological sample comprising
contacting a biological sample with an antibody, or antibody portion, of
the invention and detecting either the antibody (or antibody portion)
bound to hIL-12 or unbound antibody (or antibody portion), to thereby
detect hIL-12 in the biological sample. The antibody is directly or
indirectly labeled with a detectable substance to facilitate detection of
the bound or unbound antibody. Suitable detectable substances include
various enzymes, prosthetic groups, fluorescent materials, luminescent
materials and radioactive materials. Examples of suitable enzymes include
horseradish peroxidase, alkaline phosphatase, β-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group complexes
include streptavidin/biotin and avidin/biotin; examples of suitable
fluorescent materials include umbelliferone, fluorescein, fluorescein
isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl
chloride or phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include 125I,
131I, 35S or 3H.

[0682] Alternative to labeling the antibody, hIL-12 can be assayed in
biological fluids by a competition immunoassay utilizing rhIL-12
standards labeled with a detectable substance and an unlabeled
anti-hIL-12 antibody. In this assay, the biological sample, the labeled
rhIL-12 standards and the anti-hIL-12 antibody are combined and the
amount of labeled rhIL-12 standard bound to the unlabeled antibody is
determined. The amount of hIL-12 in the biological sample is inversely
proportional to the amount of labeled rhIL-12 standard bound to the
anti-hIL-12 antibody.

[0683] The Y61 and J695 antibodies of the invention can also be used to
detect IL-12 from species other than humans, in particular IL-12 from
primates. For example, Y61 can be used to detect IL-12 in the cynomolgus
monkey and the rhesus monkey. J695 can be used to detect IL-12 in the
cynomolgus monkey, rhesus monkey, and baboon. However, neither antibody
cross reacts with mouse or rat IL-12 (see Example 3, subsection F).

[0684] The antibodies and antibody portions of the invention are capable
of neutralizing hIL-12 activity in vitro (see Example 3) and in vivo (see
Example 4). Accordingly, the antibodies and antibody portions of the
invention can be used to inhibit IL-12 activity, e.g., in a cell culture
containing hIL-12, in human subjects or in other mammalian subjects
having IL-12 with which an antibody of the invention cross-reacts (e.g.
primates such as baboon, cynomolgus and rhesus). In a preferred
embodiment, the invention provides an isolated human antibody, or
antigen-binding portion thereof, that neutralizes the activity of human
IL-12, and at least one additional primate IL-12 selected from the group
consisting of baboon IL-12, marmoset IL-12, chimpanzee IL-12, cynomolgus
IL-12 and rhesus IL-12, but which does not neutralize the activity of the
mouse IL-12. Preferably, the IL-12 is human IL-12. For example, in a cell
culture containing, or suspected of containing hIL-12, an antibody or
antibody portion of the invention can be added to the culture medium to
inhibit hIL-12 activity in the culture.

[0685] In another embodiment, the invention provides a method for
inhibiting IL-12 activity in a subject suffering from a disorder in which
IL-12 activity is detrimental. IL-12 has been implicated in the
pathophysiology of a wide variety of disorders (Windhagen et al., (1995)
J. Exp. Med. 182: 1985-1996; Morita et al. (1998) Arthritis and
Rheumatism. 41: 306-314; Bucht et al., (1996) Clin. Exp. Immunol. 103:
347-367; Fais et al. (1994) J. Interferon Res. 14:235-238; Parronchi et
al., (1997) Am. J. Path. 150:823-832; Monteleone et al., (1997)
Gastroenterology. 112:1169-1178, and Berrebi et al., (1998) Am. J. Path
152:667-672; Parronchi et al (1997) Am. J. Path. 150:823-832). The
invention provides methods for inhibiting IL-12 activity in a subject
suffering from such a disorder, which method comprises administering to
the subject an antibody or antibody portion of the invention such that
IL-12 activity in the subject is inhibited. Preferably, the IL-12 is
human IL-12 and the subject is a human subject. Alternatively, the
subject can be a mammal expressing a IL-12 with which an antibody of the
invention cross-reacts. Still further the subject can be a mammal into
which has been introduced hIL-12 (e.g., by administration of hIL-12 or by
expression of an hIL-12 transgene). An antibody of the invention can be
administered to a human subject for therapeutic purposes (discussed
further below). Moreover, an antibody of the invention can be
administered to a non-human mammal expressing a IL-12 with which the
antibody cross-reacts for veterinary purposes or as an animal model of
human disease. Regarding the latter, such animal models may be useful for
evaluating the therapeutic efficacy of antibodies of the invention (e.g.,
testing of dosages and time courses of administration).

[0686] As used herein, the phrase "a disorder in which IL-12 activity is
detrimental" is intended to include diseases and other disorders in which
the presence of IL-12 in a subject suffering from the disorder has been
shown to be or is suspected of being either responsible for the
pathophysiology of the disorder or a factor that contributes to a
worsening of the disorder. Accordingly, a disorder in which IL-12
activity is detrimental is a disorder in which inhibition of IL-12
activity is expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an increase in
the concentration of IL-12 in a biological fluid of a subject suffering
from the disorder (e.g., an increase in the concentration of IL-12 in
serum, plasma, synovial fluid, etc. of the subject), which can be
detected, for example, using an anti-IL-12 antibody as described above.
There are numerous examples of disorders in which IL-12 activity is
detrimental. In one embodiment, the antibodies or antigen binding
portions thereof, can be used in therapy to treat the diseases or
disorders described herein. In another embodiment, the antibodies or
antigen binding portions thereof, can be used for the manufacture of a
medicine for treating the diseases or disorders described herein. The use
of the antibodies and antibody portions of the invention in the treatment
of a few non-limiting specific disorders is discussed further below:

A. Rheumatoid Arthritis:

[0687] Interleukin-12 has been implicated in playing a role in
inflammatory diseases such as rheumatoid arthritis. Inducible IL-12p40
message has been detected in synovia from rheumatoid arthritis patients
and IL-12 has been shown to be present in the synovial fluids from
patients with rheumatoid arthritis (see e.g., Morita et al., (1998)
Arthritis and Rheumatism 41: 306-314). IL-12 positive cells have been
found to be present in the sublining layer of the rheumatoid arthritis
synovium. The human antibodies, and antibody portions of the invention
can be used to treat, for example, rheumatoid arthritis, juvenile
rheumatoid arthritis, Lyme arthritis, rheumatoid spondylitis,
osteoarthritis and gouty arthritis. Typically, the antibody, or antibody
portion, is administered systemically, although for certain disorders,
local administration of the antibody or antibody portion may be
beneficial. An antibody, or antibody portion, of the invention also can
be administered with one or more additional therapeutic agents useful in
the treatment of autoimmune diseases.

[0688] In the collagen induced arthritis (CIA) murine model for rheumatoid
arthritis, treatment of mice with an anti-IL-12 mAb (rat anti-mouse IL-12
monoclonal antibody, C17.15) prior to arthritis profoundly suppressed the
onset, and reduced the incidence and severity of disease. Treatment with
the anti-IL-12 mAb early after onset of arthritis reduced severity, but
later treatment of the mice with the anti-IL-12 mAb after the onset of
disease had minimal effect on disease severity.

[0690] Interleukin-12 has been implicated as a key mediator of multiple
sclerosis. Expression of the inducible IL-12 p40 message or IL-12 itself
can be demonstrated in lesions of patients with multiple sclerosis
(Windhagen et al., (1995) J. Exp. Med. 182: 1985-1996, Drulovic et al.,
(1997) J. Neurol. Sci. 147: 145-150). Chronic progressive patients with
multiple sclerosis have elevated circulating levels of IL-12.
Investigations with T-cells and antigen presenting cells (APCs) from
patients with multiple sclerosis revealed a self-perpetuating series of
immune interactions as the basis of progressive multiple sclerosis
leading to a Th1-type immune response. Increased secretion of IFN-γ
from the T cells led to increased IL-12 production by APCs, which
perpetuated the cycle leading to a chronic state of a Th1-type immune
activation and disease (Balashov et al., (1997) Proc. Natl. Acad. Sci.
94: 599-603). The role of IL-12 in multiple sclerosis has been
investigated using mouse and rat experimental allergic encephalomyelitis
(EAE) models of multiple sclerosis. In a relapsing-remitting EAE model of
multiple sclerosis in mice, pretreatment with anti-IL-12 mAb delayed
paralysis and reduced clinical scores. Treatment with anti-IL-12 mAb at
the peak of paralysis or during the subsequent remission period reduced
clinical scores. Accordingly, the antibodies or antigen binding portions
thereof of the invention may serve to alleviate symptoms associated with
multiple sclerosis in humans.

D. Insulin-Dependent Diabetes Mellitus

[0691] Interleukin-12 has been implicated as an important mediator of
insulin-dependent diabetes mellitus (IDDM). IDDM was induced in NOD mice
by administration of IL-12, and anti-IL-12 antibodies were protective in
an adoptive transfer model of IDDM. Early onset IDDM patients often
experience a so-called "honeymoon period" during which some residual
islet cell function is maintained. These residual islet cells produce
insulin and regulate blood glucose levels better than administered
insulin. Treatment of these early onset patients with an anti-IL-12
antibody may prevent further destruction of islet cells, thereby
maintaining an endogenous source of insulin.

[0697] Antibodies to hIL-12 were isolated by screening three separate scFv
phage display libraries prepared using human VL and VH cDNAs from mRNA
derived from human tonsils (referred to as scFv 1), tonsil and peripheral
blood lymphocytes (PBL) (referred to as scFv 2), and bone marrow-derived
lymphocytes (referred to as BMDL). Construction of the library and
methods for selection are described in Vaughan et al. (1996) Nature
Biotech. 14: 309-314.

[0698] The libraries were screened using the antigens, human IL-12 p70
subunit, human IL-12 p40 subunit, chimaeric IL-12 (mouse p40/human p35),
mouse IL-12, biotinylated human IL-12 and biotinylated chimaeric IL-12.
IL-12 specific antibodies were selected by coating the antigen onto
immunotubes using standard procedures (Marks et al., (1991) J. Mol. Biol.
222: 581-597). The scFv library 2 was screened using either IL-12, or
biotinylated-IL-12, and generated a significant number of IL-12 specific
binders. Five different clonotypes were selected, determined by BstN1
enzymatic digestion patterns, and confirmed by DNA sequencing. The main
clonotypes were VHDP58NVLDPL11, VHDP77NVLDPK31, VHDP47/VL and
VHDP77/VLDPK31, all of which recognized the p40 subunit of IL-12.

[0699] Screening of the BMDL library with IL-12 p70 generated 3 different
clonotypes. Two of these were found to be cross-reactive clones. The
dominant clone was sequenced and consisted of VHDP35/VLDP. This clone
recognizes the p40 subunit of IL-12. Screening of the scFv library 1,
using IL-12 p70, did not produce specific IL-12 antibodies.

[0700] In order to identify IL-12 antibodies which preferentially bind to
the p70 heterodimer or the p35 subunit of IL-12, rather than the p40
subunit, the combined scFv 1+2 library, and the BMDL library were used.
To select IL-12 antibodies that recognized the p70 heterodimer or p35
subunit, phage libraries were preincubated and selected in the presence
of free p40. Sequencing of isolated clones revealed 9 different antibody
lineages. Subunit preferences were further analyzed by `micro-Friguet`
titration. The supernatant containing scFv was titrated on
biotin-captured IL-12 in an ELISA and the ED50 determined. The
concentration of scFv producing 50% ED was preincubated with increasing
concentrations of free p70 or p40 (inhibitors). A decrease in the ELISA
signal on biotin-IL-12 coated plates was measured and plotted against the
concentration of free p70 or p40. This provided the IC50 for each
clone with respect to p70 and p40. If the titrations for both subunits
overlaps, then the scFv binds to both p40 and p70. Any variation from
this gives the degree of preference of p70 over p40.

B. Affinity Maturation of Antibody Lineage Specific for IL-12 (Joe 9)

[0701] The clones were tested for their ability to inhibit IL-12 binding
to its receptor in an IL-12 receptor binding assay (referred to as RBA),
and for their ability to inhibit IL-12 induced proliferation of PHA
stimulated human blast cells (PHA assay), described in Example 3. Clone
Joe 9 had the lowest IC50 value in both the RBA and the PHA assay,
with an IC50 value of 1×10-6 M in both assays. In
addition the heavy chain variable region (VH) of Joe 9 had the least
number of changes compared to the closest germline sequence COS-3,
identified from the VBASE database. Table 1 (see Appendix A) shows the
VH3 family of germline sequences, of which COS-3 is a member, as
well as members of V.sub.λ1 family of germline sequences.
Therefore, Joe 9 was selected for affinity maturation. The amino acids
sequences of VH and VL of the Joe9 wild type (Joe9 wt) antibody are shown
in FIG. 1A-1D.

[0702] In order to increase the affinity of Joe 9, various mutations of
the complementarity determining region 3 (CDR3) of both the heavy and
light chains were made. The CDR3 variants were created by site-directed
PCR mutagenesis using degenerate oligonucleotides specific for either the
heavy chain CDR3 (referred to as "H3") or the light chain CDR3 (referred
to as "L3"), with an average of three base substitutions in each CDR3
(referred to as "spike"). PCR mutagenesis of the heavy chain CDR3 was
performed using the degenerate heavy chain oligonucleotide containing a
random mixture of all four nucleotides,
5'TGTCCCTTGGCCCCA(G)(T)(A)(G)(T)(C)(A)(T)(A)(G)(C)(T)(C)(C)(C)(A)(C)(T)
GGTCGTACAGTAATA 3' (SEQ ID NO: 580), and oligonucleotide pUC Reverse Tag
GAC ACC TCG ATC AGC GGA TAA CAA TTTCAC ACA GG (SEQ ID NO: 581) to
generate a repertoire of heavy chain CDR3 mutants. The parent light chain
was amplified using Joe 9 reverse oligonucleotide (5'TGG GGC CAA GGG
ACA3' (SEQ ID NO:582) and the fdteteseq 24+21 oligonucleotide (5'-ATT CGT
CCT ATA CCG TTC TAC TTT GTC GTC TTT CCA GAC GTT AGT-3' (SEQ ID NO: 583).

[0704] Heavy chain CDR3 mutants were selected using 1 nM biotinylated
IL-12, and washed for 1 h at room temperature in PBS containing free
IL-12 or p40 at a concentration of 7 nM. Clones were analyzed by phage
ELISA and those that bound to IL-12 were tested in BIAcore kinetic
binding studies using a low density IL-12 chip (see procedure for BIAcore
analysis in Example 5). Generally, BIAcore analysis measures real-time
binding interactions between ligand (recombinant human IL-12 immobilized
on a biosensor matrix) and analyte (antibodies in solution) by surface
plasmon resonance (SPR) using the BIAcore system (Pharmacia Biosensor,
Piscataway, N.J.). The system utilizes the optical properties of SPR to
detect alterations in protein concentrations within a dextran biosensor
matrix. Proteins are covalently bound to the dextran matrix at known
concentrations. Antibodies are injected through the dextran matrix and
specific binding between injected antibodies and immobilized ligand
results in an increased matrix protein concentration and resultant change
in the SPR signal. These changes in SPR signal are recorded as resonance
units (RU) and are displayed with respect to time along the y-axis of a
sensorgram. To determine the off rate (koff), on rate (kon),
association rate (Ka) and dissociation rate (Kd) constants, BIAcore
kinetic evaluation software (version 2.1) was used. Clones that
demonstrated an improvement in the koff rate were analyzed by
neutralization assays which included inhibition by antibody of IL-12
binding to its receptor (RBA assay), inhibition of IL-12-induced
proliferation in PHA stimulated human blast cells (PHA assay), and
inhibition of IL-12-induced interferon gamma production by human blast
cells (IFN gamma assay). A summary of the dissociation rates and/or
IC50 values from neutralization assays of heavy chain CDR3 spiked
clones 70-1 through 70-13 is presented in Table 2 (see Appendix A). Clone
70-1 displayed a koff rate that was better than the parent Joe 9
clone, and had the lowest IC50 value of 2.0×10-7 M.
Therefore clone 70-1 was selected for conversion to complete IgG1.

[0705] Light chain CDR3 mutants were selected using 1 nM biotin-IL-12 and
washed with PBS containing 7 nM free p40. Clones were screened in phage
ELISA and those that bound to IL-12 were tested in BIAcore binding
analysis using low density IL-12 chips. Clones that displayed an off rate
which was better than the parent Joe 9 clone were tested in
neutralization assays which measured either, inhibition of IL-12 receptor
binding, or inhibition of PHA blast cell proliferation. A summary of the
dissociation rates and/or IC50 values from neutralization assays of
light chain CDR3 mutant clones, 78-34 through 79-1, is presented in Table
2 (see Appendix A).

[0706] Based on the koff rate, clones 78-34 and 78-35 displayed an
improved koff rate compared to the parent Joe 9. Both of these
clones were selected for combination analysis with heavy chain mutants.

C. Combination Clones

[0707] Mutant light and heavy chain clones that exhibited the best binding
characteristics were used for combination and assembly of scFvs. Mutant
clones with improved potency characteristics were combined by PCR overlap
extension and pull-through of the mutated VH and VL segments as described
above. Clones 101-14 through 26-1, shown in Table 2 (see Appendix A),
were produced from the combination of heavy chain mutants (70-2, 70-13
and 70-1) with light chain mutants (78-34, 78-35 and 79-1). The koff
rates and/or IC50 values from neutralization assays for these clones
are presented in Table 2.

[0708] BIAcore binding analysis identified clone 101-11, produced from the
combination of the heavy chain CDR3 mutant clone 70-1 with the light
chain CDR3 mutant clone 78-34, as having an off rate of 0.0045 si. This
koff rate was a significant improvement compared to the koff
rates for either the heavy chain CDR3 mutant clone 70-1 (0.0134s-1),
or for the light chain CDR3 mutant clone 78-34 (0.0164s-1) alone.
Furthermore, clone 101-11 showed a significant improvement in
neutralization assays. Accordingly, clone 101-11 was selected for
affinity maturation as described below.

D. Affinity Maturation of Clone 101-11

[0709] Further affinity maturation of clone 101-11 consisted of repeat
cycles of PCR mutagenesis of both the heavy and light chain CDR3s of
101-11 using spiked oligonucleotide primers. The clones were selected
with decreasing concentrations of biotinylated IL-12 (bio-IL-12). The
binding characteristics of the mutated clones was assessed by BIAcore
binding analysis and RBA, PHA neutralization assays. The koff rates
and/or IC50 values for clones 136-9 through 170-25 are presented in
Table 2 (see Appendix A). Clone 103-14 demonstrated an improved IC50
value in both the receptor binding assay and the PHA blast assay. Clone
103-14 also demonstrated a low koff rate, and accordingly was
selected for further affinity maturation.

[0711] Randomized mutagenesis of all three light chain CDRs (referred to
as L3.1, L3.2, and L3.3) of clone 103-14 was performed. The heavy chain
CDR3 (referred to as H3) of clone 103-14 was not mutated. Four randomized
libraries based on clone 103-14 (H3 and L3.1, L3.2 & L3.3) were
constructed and subjected to a large variety of selection conditions that
involved using limiting antigen concentration and the presence or absence
of excess free antigen (p40 and p70). The outputs from selections (clones
73-B1 through 99-G11) were screened primarily by BIAcore, and on occasion
with RBA and are shown in Table 2 (see Appendix A).

[0712] Random mutagenesis of the light chain CDR of 103-14 generated clone
Y61, which exhibited a significant improvement in IC50 value
compared to the parent clone 103-14. Y61 was selected for conversion to a
whole IgG1. Whole Y61-IgG1 has an IC50 value of approximately 130 pM
determined by the PHA assay. The IC50 value was not affected by a 50
fold molar excess of free p40, demonstrating that free p40 did not
cross-react with Y61 anti-IL-12 antibody to thereby decrease the antibody
binding to the heterodimer. The full length sequences of Y61 heavy chain
variable region and light chain variable region are shown below.

[0715] Typically selection of recombinant antibodies with improved
affinities can be carried out using phage display methods. This is
accomplished by randomly mutating combinations of CDR residues to
generate large libraries containing single-chain antibodies of different
sequences. Typically, antibodies with improved affinities are selected
based on their ability to reach an equilibrium in an antibody-antigen
reaction. However, when Y61 scFV was expressed on phage surface and
incubated with IL-12, selection conditions could not be found that would
allow the system to reach normal antibody-antigen equilibrium. The
scFV-phage remained bound to IL-12, presumably due to a non-specific
interaction, since purified Y61 scFv exhibits normal dissociation
kinetics. Since the usual methods of phage-display affinity maturation to
Y61 (i.e. library generation and selections by mutagenesis of multiple
CDR residues) could not be utilized, a new strategy was developed in
which individual CDR positions were mutated.

[0716] This strategy involves selection of appropriate CDR positions for
mutation and is based on identification and selection of amino acids that
are preferred selective mutagenesis positions, contact positions, and/or
hypermutation positions. Contact positions are defined as residues that
have a high probability of contact with an antigen when the antigen
interacts with the antibody, while hypermutation positions are defined as
residues considered to have a high probability for somatic hypermutation
during in vivo affinity maturation of the antibody. Preferred selective
mutagenesis positions are CDR positions that are both contact and
hypermutation positions. The Y61 antibody was already optimized in the
CDR3 regions using the procedure described in Example 1, therefore it was
difficult to further improve the area which lies at the center of the
antibody binding site using phage-display selection methods. Greater
improvements in activity were obtained by mutation of potential contact
positions outside the CDR3 regions by either removing a detrimental
antigen-antibody contact or, engineering a new contact.

[0717] Amino acids residues of Y61 which were considered contact points
with antigen, and those CDR positions which are sites of somatic
hypermutations during in vivo affinity maturation, are shown in Table 3
(see Appendix A). For Y61 affinity maturation, 15 residues outside CDR3,
3 residues within the L3 loop, and 5 residues in the H3 loop were
selected for PCR mutagenesis.

[0718] Y61 scFv gene was cloned into the pUC119(Sfi) plasmid vector for
mutagenesis. Oligonucleotides were designed and synthesized with
randomized codons to mutate each selected position. Following PCR
mutagenesis, a small number of clones (˜24) were sequenced and
expressed in a host cell, for example, in a bacterial, yeast or mammalian
host cell. The expressed antibody was purified and the koff measured
using the BIAcore system. Clones with improved off-rates, as compared to
Y61, were then tested in neutralization assays. This procedure was
repeated for other CDR positions. Individual mutations shown to have
improved neutralization activity were combined to generate an antibody
with even greater neutralization potency.

[0719] The Y61 CDR positions that were mutated in order to improve
neutralization potency, and the respective amino-acid substitutions at
each position are shown in FIGS. 2A-2H. Off-rates, as determined by
BIAcore analysis, are given. These off rates are also shown in the
histograms to the right of each table.

[0720] Results of these substitutions at positions H30, H32, H33, H50,
H53, H54, H58, H95, H97, H101, L50, L92, L93, demonstrated that all
amino-acid substitutions examined resulted in antibodies with poorer
off-rates than Y61. At positions H52, L32, and L50, only a one amino acid
substitution was found to improve the off-rate of Y61, all other changes
adversely affected activity. For L50, this single Gly-Tyr change
significantly (5-10 times) improved the neutralization potency of Y61.
The results demonstrated the importance of these positions to Y61
activity, and suggest that in most cases phage-display was able to select
for the optimal residues. However, at positions H31, H56, L30, and L94,
several substitutions were found to improve Y61 off-rate, suggesting that
these positions were also important for antigen binding, although the
phage display approach did not allow selection of the optimal residues.

[0721] Selective mutation of contact and hypermutation positions of Y61
identified amino acid residue L50 in the light chain CDR2, and residue
L94 of the light chain CDR3, which improved the neutralization ability of
Y61. A combination of these mutations produced an additive effect,
generating an antibody, J695, that exhibited a significant increase in
neutralization ability. The full length sequence of J695 heavy and light
chain variable region sequences is shown below.

[0724] A summary of the heavy and light chain variable region sequence
alignments showing the lineage development of clones that were on the
path from Joe9 to J695 is shown in FIGS. 1A-1D. The CDRs and residue
numbering are according to Kabat.

Example 3

Functional Activity of Anti-hIL-12 Antibodies

[0725] To examine the functional activity of the human anti-human IL-12
antibodies of the invention, the antibodies were used in several assays
that measure the ability of an antibody to inhibit IL-12 activity.

A. Preparation of Human PHA-Activated Lymphoblasts

[0726] Human peripheral blood mononuclear cells (PBMC) were isolated from
a leukopac collected from a healthy donor by Ficoll-Hypaque gradient
centrifugation for 45 minutes at 1500 rpm as described in Current
Protocols in Immunology, Unit 7.1. PBMC at the interface of the aqueous
blood solution and the lymphocyte separation medium were collected and
washed three times with phosphate-buffered saline (PBS) by centrifugation
for 15 minutes at 1500 rpm to remove Ficoll-Paque particles.

[0727] The PBMC were then activated to form lymphoblasts as described in
Current Protocols in Immunology, Unit 6.16. The washed PBMC were
resuspended at 0.5-1×106 cells/ml in RPMI complete medium
(RPMI 1640 medium, 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100
μg/ml streptomycin), supplemented with 0.2% (v/v) PHA-P (Difco,
Detroit, Mich.) and cultured for four days at 37° C. in a 5%
CO2 atmosphere. After four days, cell cultures were split 1:1 by
volume in RPMI complete medium, plus 0.2% (v/v) PHA-P and 50 U/ml
recombinant human IL-2. Recombinant human IL-2 was produced by
transfection of an expression vector carrying the human IL-2 cDNA into
COS cells (see Kaufman et al., (1991) Nucleic Acids Res. 19, 4484-4490),
and purified as described in PCT/US96/01382. Cell cultures were then
incubated for an additional one to three days. PHA blast cells were
harvested, washed twice with RPMI complete medium and frozen in 95% FBS,
5% DMSO at 10×106 cells/ml.

[0728] PHA blast cells to be used for the IL-12 receptor binding assay
(see section B) were collected after one day culture in the presence of
IL-2, whereas PHA blast cells to be used for the PHA blast proliferation
assay (see section C) and the interferon-gamma induction assay (see
section D) were collected after three day culture in the presence of
IL-2.

B. IL-12 Receptor Binding Assay

[0729] The ability of anti-IL-12 antibodies to inhibit binding of
radiolabelled IL-12 to IL-12 receptors on PHA blasts were analyzed as
follows. Various concentrations of anti-IL-12 antibody were preincubated
for 1 hour at 37° C. with 50-100 pM 125I-hIL-12 (iodinated
hIL-12 was prepared using the Bolton-Hunter labeling method to a specific
activity of 20-40mCi/mg from NEN-Dupont) in binding buffer (RPMI 1640, 5%
FBS, 25 mM Hepes pH 7.4). PHA blast cells isolated as described above,
were washed once and resuspended in binding buffer to a cell density of
2×107 cells/ml. PHA blasts (1×106 cells) were added
to the antibody 125I-hIL-12 mixture and incubated for two hours at
room temperature. Cell bound radioactivity was separated from free
125I-hIL-12 by centrifugation of the assay mixture for 30 seconds at
room temperature, aspiration of the liquid and a wash with 0.1 ml binding
buffer, followed by centrifugation at 4° C. for 4 min at
10,000×g. The cell pellet was examined for cell bound radioactivity
using a gamma counter. Total binding was determined in the absence of
antibody and non-specific binding was determined by inclusion of 25 nM
unlabeled IL-12 in the assay. Incubations were carried out in duplicate.

[0730] In the IL-12 receptor binding assay using the Y61 and J695 human
anti-IL-12 antibodies, both antibodies demonstrated a comparable
inhibition of IL-12 receptor binding. Y61 inhibited IL-12 receptor
binding with an IC50 value of approximately 1.6×10-11 M,
while J695 had an IC50 value of approximately 1.1×10-11
M.

C. Human PHA Blast Proliferation Assay

[0731] Anti-IL-12 antibodies were evaluated for their ability to inhibit
PHA blast proliferation (which proliferation is stimulated by IL-12).
Serial dilutions of anti-IL-12 antibody were preincubated for 1 hour at
37° C., 5% CO2 with 230 pg/ml hIL-12 in 100 ml RPMI complete
medium in a microtiter plate (U-bottom, 96-well, Costar, Cambridge,
Mass.). PHA blast cells isolated as described above, were washed once and
resuspended in RPMI complete medium to a cell density of 3×105
cells/ml. PHA blasts (100 ml, 3×104 cells) were added to the
antibody/hIL-12 mixture, incubated for 3 days at 37° C., 5%
CO2 and labeled for 4-6 hours with 0.5 mCi/well (3H)-Thymidine
(Amersham, Arlington Heights, Ill.). The culture contents were harvested
onto glass fiber filters by means of a cell harvester (Tomtec, Orange,
Conn.) and (3H)-Thymidine incorporation into cellular DNA was measured by
liquid scintillation counting. All samples were assayed in duplicate.

[0732] The results of neutralization in the presence of varying
concentrations of p70:p40 (i.e. the ratio of IL-12 heterodimer to free
p40 subunit) is shown in Table 4 (see Appendix A).

[0733] Analysis of the Y61 human anti-IL-12 antibody in the PHA blast
proliferation assay demonstrated that the antibody inhibited PHA blast
proliferation with an IC50 value of approximately
1.8×10-10 M in the presence of IL-12 p70 alone, without any
excess p40 (p70:p40 ratio of 1:0). In the presence of a 50-fold excess of
free p40 (p70:p40 at a ratio of 1:50), the Y61 antibody inhibited PHA
blast proliferation with an IC50 value of approximately
1.8×10-10 M. This result demonstrates that the ability of Y61
to inhibit blast proliferation is not compromised by the presence of
excess p40.

[0734] The human anti-IL-12 antibody, J695 inhibited PHA blast
proliferation with an IC50 value of approximately
1.0×10-11 M in the presence of p70:p40 at a ratio of 1:0. In
the presence of a p70:p40 ratio of 1:50, this antibody inhibited PHA
blast proliferation with an IC50 value of approximately
5.8±2.8×10-12 M (n=2), demonstrating that the excess p40
had only a slight inhibitory effect on the antibody. Overall results
demonstrate the improved neutralization activity of J695 in comparison
with Y61 due to the mutations at L50 and L94.

D. Interferon-Gamma Induction Assay

[0735] The ability of anti-IL-12 antibodies to inhibit the production of
IFNγ by PHA blasts (which production is stimulated by IL-12) was
analyzed as follows. Various concentrations of anti-IL-12 antibody were
preincubated for 1 hour at 37° C., 5% CO2 with 200-400 pg/ml
hIL-12 in 100 ml RPMI complete medium in a microtiter plate (U-bottom,
96-well, Costar). PHA blast cells isolated as described above, were
washed once and resuspended in RPMI complete medium to a cell density of
1×07 cells/ml. PHA blasts (100 μl of 1×106 cells)
were added to the antibody/hIL-12 mixture and incubated for 18 hours at
37° C. and 5% CO2. After incubation, 150 μl of cell free
supernatant was withdrawn from each well and the level of human
IFNγ produced was measured by ELISA (Endogen Interferon gamma
ELISA, Endogen, Cambridge, Mass.). Each supernatant was assayed in
duplicate.

[0736] Analysis of human anti-hIL-12 antibody, Y61 in this assay
demonstrated that Y61 inhibited human IFNγ production with an
IC50 value of approximately 1.6×10-10 M, while the human
anti-IL-12 antibody, J695, inhibited human IFNγ production with an
IC50 value of approximately 5.0±2.3×10-12 M (n=3). The
result demonstrates the substantial improvement in the affinity of J695
as a result of the modifications at L50 and L94.

E. Induction of Non-Human IL-12 from Isolated PBMC

[0737] To examine the cross-reactivity of the human anti-hIL-12 antibodies
with IL-12 from other species, non-human IL-12 was produced as follows.
PBMC were separated from fresh heparinized blood by density gradient
centrifugation as described above using lymphoprep (Nycomed, Oslo,
Norway) for cynomolgus monkey, baboon, and dog, PBMC, Accu-paque
(Accurate Chemical & Sci. Corp., Westbury, N.Y.) for dog PBMC or
Lympholyte-rat (Accurate Chemical & Sci. Corp., Westbury, N.Y.) for rat
PBMC.

[0741] Serial dilutions of anti-IL-12 antibody were preincubated for 1
hour at 37° C., 5% CO2 with 40 pg/ml murine IL-12 in 100 ml
RPMI complete medium plus PME in a microtiter plate (U-bottom, 96-well,
Costar). 2D6 cells were washed once and resuspended in RPMI complete
medium containinγ PME to a cell density of 1×105
cells/ml. 2D6 cells (100 μl, 1×104 cells) were added to the
antibody/hIL-12 mixture, incubated for 3 days at 37° C., 5%
CO2 and labeled for 4-6 hours with 0.5 mCi/well (3H)-Thymidine. The
culture contents were harvested and counted by liquid scintillation
counting. All samples were assayed in duplicate.

G. Species Cross-Reactivity of J695 with Non-Human IL-12

[0742] Species cross-reactivity of J695 with non-human IL-12 was analyzed
using PBMC's isolated from several non-human species. The presence of
non-human IL-12 activity in the rat, dog, cynomolgus and baboon PBMC
supernatants was confirmed using several bioassays described above, such
as the murine 2D6 cell proliferation assay, the human PHA blast
proliferation assay and the interferon-gamma induction assay by blocking
the non-human PBMC induced responses with rabbit and/or sheep polyclonal
antibodies to murine and/or human IL-12. Cross-reactivity of the human
anti-hIL-12 antibodies Y61 and J695 with non-human IL-12 in PBMC
supernatants or purified murine and rhesus IL-12 was then assessed in the
same bioassay(s) by determining the J695 antibody concentration at which
50% inhibition of the response was observed. The species cross-reactivity
results are summarized in Table 5. The results demonstrate that Y61 and
J695 are each able to recognize IL-12 from monkeys (e.g, cynomolgus and
rhesus IL-12 for Y61, and cynomolgus, rhesus and baboon for J695) and
that J695 is approximately 35 fold less active on dog IL-12; neither Y61
nor J695 cross reacts with mouse or rat IL-12.

[0745] The results demonstrated that J695 binding to immobilized human
IL-12 was blocked only by human IL-12 p70 and to a lesser extent, by
human IL-12 p40 and not by any of the other cytokines tested.

I. Binding to a Novel IL-12 Molecule

[0746] An alternative IL-12 heterodimer has been described, in which the
p35 subunit is replaced by a novel p19 molecule. P19 was identified using
3D homology searching for IL-6/IL-12 family members, and is synthesized
by activated dendritic cells. P19 binds to p40 to form a p19/p40 dimer,
which has IL-12-like activity, but is not as potent as the p35/p40
heterodimer in IFNγ induction. Antibodies which recognize p40
alone, but preferably in the context of a p70 molecule (e.g., J695 and
Y61, see Example 3H) are expected to also neutralize both the p35/p40
molecules and the p19/p40 molecules.

Example 4

In Vivo Activity of Anti-hIL-12 Antibodies

[0747] The in vivo effects of IL-12 antibodies on IL-12 induced responses
were examined in a model modified from one used by Bree et al. to study
the effect of human IL-12 on peripheral hematology in cynomolgus monkey
Bree et al., (1994) Biochem Biophys Res. Comm. 204: 1150-1157. In those
previous studies, administration of human IL-12 at 1 μg/kg/day for a
period of 5 days resulted in a decrease in white blood cell count (WBC),
especially in the lymphocyte and monocyte subsets after 24 hours. A
decrease in the platelet count was observed at 72 hours. Levels of plasma
neopterin, a marker of monocyte activation in response to IFN-γ,
began to elevate at 24 hours and were the highest at 72 hours.

[0748] In the first study with human anti-hIL-12 antibodies, fifteen
healthy cynomolgus monkeys with an average weight of 5 kg, were sedated
and divided into 5 groups (n=3). Group 1 received an intravenous (IV)
administration of 10 mg/kg human intravenous immunoglobulin (IVIG, Miles,
Eckhart, Ind., purified using protein A Sepharose). Group 2 received an
intravenous administration of 1 mg/kg C8.6.2 (neutralizing mouse
anti-human IL-12 monoclonal antibody). Group 3 received an intravenous
administration of 10 mg/kg C8.6.2. Group 4 received an intravenous
administration of 1 mg/kg Y61 (human anti-human IL-12 antibody, purified
from CHO cell conditioned medium). Group 5 received an intravenous
administration of 10 mg/kg Y61.

[0749] One hour after the antibody administration all animals received a
single subcutaneous (SC) injection of human IL-12 (1 μg/kg). Blood
samples were taken at the following time points: baseline, 8, 24, 48, 96
and 216 hours, and analyzed for complete blood cell counts with
differentials and serum chemistry. Serum human IL-12, C8.6.2 antibody,
Y61 antibody, monkey IFN-gamma, monkey IL-10, monkey IL-6 and plasma
neopterin levels were also measured.

[0750] Animals treated with IL-12 plus IVIG control antibody (Group 1)
showed many of the expected hematological changes, including decreases in
WBC, platelets, lymphocyte count and monocyte count. These decreases were
not seen or were less pronounced in the animals treated with either the
C8.6.2 or Y61 antibody at 1 or 10 mg/kg (Groups 2-5).

[0751] Serum or plasma samples were analyzed by ELISA specific for monkey
IFN-gamma and monkey IL-10 (Biosource International, Camarillo, Calif.),
monkey IL-6 (Endogen) and plasma neopterin (ICN Pharmaceuticals,
Orangeburg, N.Y.). IFN-gamma, IL-10 or IL-6 were not detected in any of
the IL-12 treated animals including the control animals treated with
IL-12 plus IVIG. This was probably due to the low level exposure to IL-12
(only 1 dose of 1 μg/kg). Nevertheless, plasma neopterin levels
increased about three fold in the IL-12 plus IVIG treated animals but did
not change in all C8.6.2 or Y61 treated animals, including the lower dose
(1 mg/kg) Y61 treated animals, indicating that Y61 was effective in vivo
in blocking this sensitive response to IL-12.

[0752] In a second study, in vivo activity and pharmacodynamics (PD) of
J695 in cynomolgous monkeys were studied by administering exogenous
rhIL-12 and determining if J695 could block or reduce the responses
normally associated with rhIL-12 administration. Male cynomolgus monkeys
(n=3 per group) were administered a single dose of 0.05, 0.2, or 1.0
mg/kg J695 or 1 mg/kg intravenous immunoglobulin (IVIG) as a bolus
intravenous (IV) injection via a saphenous vein or subcutaneously (SC) in
the dorsal skin. One hour following the administration of J695 or IVIG,
all animals received a single SC dose of 1 μg/kg rhIL-12 in the dorsal
skin. Blood samples were collected via the femoral vein up to 28 days
after J695 administration. Serum was acquired from each blood sample and
assayed for IL-12, J695, IFN-γ, and anti-J695 antibodies by ELISA.
Neopterin was assayed by reverse-phase high performance liquid
chromatography.

[0753] The levels of neopterin, normalized with respect to the levels of
neopterin that were measured before administration of J695 or rhIL-12,
are shown in FIG. 3. To compare the suppression of neopterin between
groups, the area under the curve (AUC) normalized for neopterin levels
was calculated for each animal (Table 6). Neopterin exposure (AUC) was
suppressed in a dose-dependent manner between approximately 71 and 93% in
the IV groups and between 71 and 100% in SC groups, relative to the IVIG
control groups. These results suggest that the dose of J695 necessary for
50% inhibition of the neopterin response (ED50) was less than 0.05
mg/kg when administered by either the IV or SC route.
TABLE-US-00012
TABLE 6
Dose-Dependent Suppression of IL-12 Induced Neopterin by J695 in
Cynomolgus Monkeys
% Reduction of
AUC of Neopterin AUC
Route of dosing IVIG J695 Dose IVIG Dose Normalized Compared with
or J695 and rhIL-12 (mg/kg) (mg/kg) Neopterin Levels Control
Single IV injection -- 1.0 1745 ± 845 0
followed 1 hr later by a 0.05 -- 502 ± 135 71.3
dose of 1 μg/kg human 0.2 -- 199 ± 316 88.6
IL-12 given SC 1.0 -- 128 ± 292 92.7
Single SC injection -- 1.0 1480 ± 604 0
followed 1 hour later 0.05 -- 426 ± 108 71.2
by a dose of 1 μg/kg 0.2 -- 395 ± 45.9 73.3
human IL-12 given SC 1.0 -- 0 ± 109 100

[0754] Treatment with J695 also prevented or reduced the changes in
hematology normally associated with rhIL-12 administration (leukopenia
and thrombocytopenia). At 24 hours after rhIL-12 administration
lymphocyte counts were reduced by approximately 50% when compared to
baseline values in the control IV and SC IVIG treated groups.
Administration of J695 either SC or IV at all three dose levels prevented
this reduction, resulting in lymphocyte counts at 24 hours approximately
the same as baseline values. At 48 hours after IL-12 administration,
platelet counts in the groups treated with W and SC IVIG were reduced by
approximately 25% when compared to baseline values.

[0755] An example dose schedule targeted to maintain serum levels above
the 90% effect level would be 1 mg/kg IV and SC given approximately every
other week, or 0.3 mg/kg given approximately every week, assuming slight
accumulation during repeated dosing. This study demonstrates that
antibody can be given safely to monkeys at such dosages. In independent
toxicity studies, it was further found that up to 100 mg/kg of the
antibody can be given safely to monkeys.

[0756] J695 was also effective in preventing IFN-γ production in
mice treated with a chimeric IL-12, a molecule which combines the murine
p35 subunit with the human IL-12 p40 subunit. In contrast to human IL-12
which is biologically inactive in mice, this chimeric IL-12 retains
biological function in mice, including induction of IFN-γ. In
addition, the human p40 subunit allows the molecule to be bound and
neutralized by J695. Chimeric IL-12 at a dose of 0.05 mg/kg i.p. was
administered to female C3H/HeJ mice (10/experimental group) in five daily
doses on days 0, 1, 2, 3, and 4. J695 was given on days 0, 2 and 4 at
doses of 0.05, 0.01, 0.002, 0.0004, 0.00008, and 0.000016 mg/kg i.p., 30'
prior to the IL-12 injections. The control hulgGlγ was given IP at
a dose of 0.05 mg/kg on days 0, 2, and 4. The mice were bled on day 5,
and serum IFN-γ levels were determined by ELISA. The results
demonstrated that J695 caused dose-dependent inhibition of IFN-γ
production with an ED50 of approximately 0.001 mg/kg. Collectively,
these results demonstrate that J695 is a potent inhibitor of 11-12
activity in vivo.

[0757] Real-time binding interactions between captured ligand (human
anti-rhIL-12 antibody J695, captured on a biosensor matrix) and analyte
(rhIL12 in solution) were measured by surface plasmon resonance (SPR)
using the BIAcore system (Biacore AB, Uppsala, Sweden). The system
utilizes the optical properties of SPR to detect alterations in protein
concentration within a dextran biosensor matrix. Proteins are covalently
bound to the dextran matrix at known concentrations. Antibodies are
injected through the dextran matrix and specific binding between injected
antibodies and immobilized ligand results in an increased matrix protein
concentration and resultant change in the SPR signal. These changes in
SPR signal are recorded as resonance units (RU) and are displayed with
respect to time along the y-axis of a sensorgram.

[0759] J695 was diluted in HBS running buffer (Biacore AB, Cat. No.
BR-1001-88, Uppsala, Sweden) to be captured on the matrix via goat
anti-human IgG. To determine the capacity of rhIL12-specific antibodies
to bind immobilized goat anti-human IgG, a binding assay was conducted as
follows. Aliquots of J695 (25 μg/ml; 25 μl aliquots) were injected
through the goat anti-human IgG polyclonal antibody coupled dextran
matrix at a flow rate of 5 μl/min. Before injection of the protein and
immediately afterward, HBS buffer alone flowed through each flow cell.
The net difference in signal between the baseline and the point
corresponding to approximately 30 seconds after completion of J695
injection was taken to represent the amount of IgG1 J695 bound
(approximately 1200 RU's). Direct rhIL12 specific antibody binding to
soluble rhIL12 was measured. Cytokines were diluted in HBS running buffer
and 50 μl aliquots were injected through the immobilized protein
matrices at a flow rate of 5 μl/min. The concentrations of rhIL-12
employed were 10, 20, 25, 40, 50, 80, 100, 150 and 200 nM. Prior to
injection of rhIL-12, and immediately afterwards, HBS buffer alone flowed
through each flow cell. The net difference in baseline signal and signal
after completion of cytokine injection was taken to represent the binding
value of the particular sample. Biosensor matrices were regenerated using
100 mM HCl before injection of the next sample. To determine the
dissociation constant (off-rate), association constant (on-rate), BIAcore
kinetic evaluation software (version 2.1) was used.

[0762] There was a small difference between the calculated apparent
constant (Kd) for the interaction between CHO derived J695
(Kd=1.34-10M-1) and COS derived J695 (Kd=9.74×10-11
M-1) antibodies. The apparent dissociation constant (Kd) between
J695 and rhIL12 was estimated from the observed rate constants by the
formula: Kd=off-rate/on-rate.

[0763] To determine the apparent association and dissociation rate
constant for the interaction between J695 and rhIL-12, several binding
reactions were performed using a fixed amount of J695 (2 μg/ml) and
varying concentrations of rhIL-12. Real-time binding interaction
sensorgrams between captured J695 and soluble rhIL12 showed that both
forms of antibody were very similar for both the association and
dissociation phase.

[0764] To further evaluate the capacity of captured IgG1 J695 mAb to bind
soluble recombinant cytokine, a direct BIAcore method was used. In this
method, goat anti-human IgG (25 μg/ml) coupled carboxymethyl dextran
sensor surface was coated with IgG1 J695 (2 μg/ml) and recombinant
cytokine was then added. When soluble rhIL12 was injected across a
biosensor surface captured with CHO or COS derived IgG1 J695, the amount
of signal increased as the concentration of cytokine in the solution
increased. No binding was observed with rmIL12 (R&D Systems, Cat. No.
419-ML, Minneapolis, Minn.) or rh IL12 any concentration tested up to
1000 nM. These results support the conclusion that IgG1 J695 antibodies
recognize a distinct determinant on rhIL-12.

[0767] BIAcore technology was used to measure the binding of soluble
rhIL-12 to solid phase captured J695. A goat anti-human IgG antibody was
immobilized on the biosensor chips, then a fixed amount of J695 was
injected and captured on the surface. Varying concentrations of rhIL-12
were applied, and the binding of IL-12 at different concentrations to
J695 was measured as a function of time. Apparent dissociation and
association rate constants were calculated, assuming zero-order
dissociation and first order association kinetics, as well as a simple
one-to-one molecular interaction between J695 and IL-12. Three
independent experiments were performed, and the values shown are averages
for the three experiments. From these measurements, the apparent
dissociation (kd) and association (ka) rate constants were
derived and used to calculate a Kd value for the interaction (see
Table 10). The results indicated that J695 has a high affinity for
rhIL-12.
TABLE-US-00016
TABLE 10
Kinetic Parameters for the Interaction Between J695 and Human IL-12
Kinetic Parameter Value
kd 3.71 ± 0.40 × 10-5 s-1
ka 3.81 ± 0.48 × 105 M-1s-1
kd 9.74 × 10-11 M (14 ng/mL)

Example 7

Characteristics and Neutralization Activity of C17.15, a Rat Monoclonal
Antibody to Murine Interleukin-12

[0768] To assess the relevance of IL-12 treatment studies in mouse models
of inflammation and autoimmunity using monoclonal antibodies specific for
murine IL-12 to similar approaches in human disease, the interaction of
C17.15, a rat anti-murine IL-12 monoclonal antibody with murine IL-12,
was examined. The ability of C17.15 to neutralize murine IL-12 activity
in a PHA blast proliferation assay, and to block murine IL-12 binding to
cell surface receptors, was assessed, as were the kinetics of the
C17.15-murine IL-12 binding interaction.

[0770] The ability of C17.15 to inhibit the binding of murine IL-12 to
cellular receptors was also measured. Serial dilutions of C17.15 were
pre-incubated for 1 hr at 37° C. with 100 pM [125I]-murine
IL-12 in binding buffer. The 2D6 cells (2×106) were added to
the antibody/[125I]-murine IL-12 mixture and incubated for 2 hours
at room temperature. Cell-bound radioactivity was separated from free
[125I]-IL-12, and the remaining cell-bound radioactivity was
determined. Total binding of the labeled murine IL-12 to receptors on 2D6
cells was determined in the absence of antibody, and non-specific binding
was determined by the inclusion of 25 nM unlabelled murine IL-12 in the
assay. Specific binding was calculated as the total binding minus the
non-specific binding. Incubations were carried out in duplicate. The
results showed that C17.15 has an IC50 (M) of 1.5×10-10
for inhibition of binding of murine IL-12 to cellular receptors.

[0771] The affinity of C17.15 for recombinant murine IL-12 was assessed by
biomolecular interaction analysis. A goat anti-rat IgG antibody was
immobilized on the biosensor chips, followed by an injection of a fixed
amount of the C17.15 antibody, resulting in capture of C17.15 on the
surface of the chip. Varying concentrations of recombinant murine IL-12
were applied to the C17.15 surface, and the binding of murine IL-12 to
the immobilized C17.15 was measured as a function of time. Apparent
dissociation and association rate constants were calculated, assuming a
zero order dissociation and first order association kinetics as well as a
simple one to one molecular interaction between the immobilized C17.15
and murine IL-12. From these measurements, the apparent dissociation
(kd, off-rate) and association (ka, on-rate) rate constants
were calculated. These results were used to calculate a Kd value for
the interaction. An on-rate of 3.8×105 M-1s-1, an
off-rate of 1.84×104 s-1, and a Kd of
4.8×10-10 was observed for the recombinant murine IL-12-C17.15
interaction.

[0772] The observed activities of C17.15 in neutralizing murine IL-12
activity and binding to cell surface receptors, as well as the kinetics
of binding of C17.15 to murine IL-12 correlate with similar measurements
for the J695-rhIL-12 interaction. This indicates that the modes of action
of the rat anti-mouse IL-12 antibody C17.15 and anti-human IL-12 antibody
J695 are nearly identical based upon on-rate, off-rate, Kd,
IC50, and the PHA blast assay. Therefore, C17.15 was used as a
homologous antibody to J695 in murine models of inflammation and
autoimmune disease to study the effects of IL-12 blockade on the
initiation or progression of disease in these model animals (see Example
8).

Example 8

Treatment of Autoimmune or Inflammation-Based Diseases in Mice by
α-Murine IL-12 Antibody Administration

A. Suppression of Collagen-Induced Arthritis in Mice by the α-Il-12
Antibody C17.15

[0773] A correlation between IL-12 levels and rheumatoid arthritis (RA)
has been demonstrated. For example, elevated levels of IL-12 p70 have
been detected in the synovia of RA patients compared with healthy
controls (Morita et al (1998) Arthritis and Rheumatism. 41: 306-314).
Therefore, the ability of C17.15, a rat anti-mouse IL-12 antibody, to
suppress collagen-induced arthritis in mice was assessed.

[0774] Male DBA/1 mice (10/group) were immunized with type II collagen on
Day 0 and treated with C17.15, or control rat IgG, at 10 mg/kg
intraperitoneally on alternate days from Day--1 (1 day prior to collagen
immunization) to Day 12. The animals were monitored clinically for the
development of arthritis in the paws until Day 90. The arthritis was
graded as: 0--normal; 1--arthritis localized to one joint; 2--more than
one joint involved but not whole paw; 3--whole paw involved; 4--deformity
of paw; 5--ankylosis of involved joints. The arthritis score of a mouse
was the sum of the arthritic grades in each individual paw of the mouse
(max=20). The results are expressed as mean ±SEM in each group.

[0775] The results, as shown in FIG. 4, indicate that an arthritic score
was measurable in the C17.15-treated mice only after day 50
post-treatment, and that the peak mean arthritic score obtained with the
C17.15-treated mice was at least 5-fold lower than that measured in the
IgG-treated mice. This demonstrated that the rat anti-mouse IL-12
antibody C17.15 prevented the development of collagen-induced arthritis
in mice.

B. Suppression of Colitis in Mice by the Rat α-Murine IL-12
Antibody C17.15

[0776] IL-12 has also been demonstrated to play a role in the
development/pathology of colitis. For example, anti-IL-12 antibodies have
been shown to suppress disease in mouse models of colitis, e.g., TNBS
induced colitis IL-2 knockout mice (Simpson et al. (1998) J. Exp. Med.
187(8): 1225-34). Similarly, anti-IL-2 antibodies have been demonstrated
to suppress colitis formation in IL-10 knock-out mice. The ability of the
rat anti-mouse IL-12 antibody, C17.15, to suppress TNBS colitis in mice
was assessed in two studies (Davidson et al. (1998) J. Immunol. 161(6):
3143-9).

[0777] In the first study, colitis was induced in pathogen free SJL mice
by the administration of a 150 mL 50% ethanol solution containing 2.0 mg
TNBS delivered via a pediatric umbilical artery catheter into the rectum.
Control animals were treated with a 150 μL 50% ethanol solution only.
A single dose of 0.75, 0.5, 0.25, or 0.1 mg C17.15 or 0.75 mg control rat
IgG2a was given intravenously via the tail vein at day 11, and the
therapeutic effect of the treatment was assessed by weighing the animals
on days 11 and 17, and histological scoring at day 17. The weight of the
mice treated with C17.15 increased within 48 hours of antibody treatment
and normalized on day 6 after treatment. The effect of treatment with
C17.15 was confirmed histologically. Further, assessments of IFN-γ
secretion by CD4.sup.+ T-cells from spleen and colon of the treated mice,
as well as IL-12 levels from spleen or colon-derived macrophages from the
treated mice were also made (see Table 12).

[0778] In the second study, the dosing was optimized and the mice were
treated with a total dose of 0.1 mg or 0.5 mg C17.15 or 0.1 mg control
IgG2a, respectively, split between days 12 and 14. It was found that the
administration of C17.15 in a single dose at the dosage of 0.1 mg/mouse
or 0.25 mg/mouse led to only partial improvement in TNBS-induced colitis
and did not result in a significant reduction in the CD4.sup.+ T cell
production of IFN-γ in vitro, but did result in a significant
decrease in secretion of IL-12, compared to untreated controls. At a
single dose of 0.5 mg/mouse or greater a response was observed. Taking
the lowest dose of antibody tested and administering it in two divided
injections (at days 12 and 14) improved the dosing regimen, indicating
that multiple low doses can be more effective than a single bolus dose.
The data obtained are shown in Table 12.
TABLE-US-00018
TABLE 12
Anti-mouse Il-12 mAb C17.15 Suppresses Established Colitis in Mice
Weight IFN-γ spleen IL-12 spleen
Disease Treatment (g) CD4.sup.+ cells macrophages
Induction Day 0 Day 11 Day 11 Day 17 (U/mL) (pg/ml)
TNBS + Ethanol Control IgG2a 16.0 15.26 3326 300
0.75 mg
TNBS + Ethanol C17.15 0.75 mg 16.0 20.21 1732 0
TNBS + Ethanol C17.15 0.5 mg 16.36 19.94 1723 0
TNBS + Ethanol C17.15 0.25 mg 16.28 17.7 3618 7
TNBS + Ethanol C17.15 0.1 mg 16.2 17.98 3489 22
Ethanol control -- 20.76 21.16 1135 0

[0779] Administration of C17.15 monoclonal anti-IL-12 in two divided doses
spaced one day apart totaling 0.1 mg/mouse or 0.05 mg/mouse led to
complete reversal of colitis as assessed by wasting and macroscopic
appearance of the colon. In addition, this dose schedule led to
significant down-regulation of lamina propria T-cell production of
IFN-γ and macrophage production of IL-12, so that the latter were
comparable to levels seen in control ethanol-treated mice without
TNBS-colitis. Thus, C17.15 administration to mouse models for TNBS
colitis reversed the progression of the disease in a dose-dependent
manner.

[0781] An α-IL-12 antibody was found to be able to inhibit the onset
of acute EAE, to suppress the disease after onset, and to decrease the
severity of relapses in mice immunized with the autoantigen, myelin basic
protein (Banerjee, S. et al. (1998) Arthritis Rheum. (1998) 41: S33). The
beneficial effects of α-IL-12 antibody treatment in the mice
persisted for over two months after stopping treatment. It has also been
demonstrated that anti-IL-12 antibodies suppress the disease in mice that
are recipients of encephalitogenic T cells by adoptive transfer (Leonard,
J. P. et al. (1995) J. Exp. Med. 181: 281-386).

Example 9

Clinical Pharmacology of J695

[0782] In a double blind, crossover study, 64 healthy, human male subjects
were administered ascending doses of J695 or placebo. Measurement of
complement fragment C3a prior to and 0.25 h after dosing did not
demonstrate activation of the complement system. CRP and fibrinogen
levels were only increased in subjects in whom symptoms of concurrent
infections were observed.

[0783] All subjects survived and the overall tolerability of J695 was very
good. In no case did treatment have to be stopped because of adverse
events (AEs). The most commonly observed AEs were headache and common
cold/bronchitis, neither of which were categorized as severe.

[0784] One of the study subjects, a 33-year-old single male, was suffering
from psoriasis guttata at the start of the study. According to the
randomized study design, this subject by chance received 5 mg/kg J695 by
SC administration. Ten days prior to administration of the antibody, the
subject showed only small discrete papular lesions on the arms and legs.
At the time of the antibody administration, the subject displayed
increased reddening, thickness of the erythematous plaques, and increased
hyperkaratosis. One week after J695 administration, the subject reported
an improvement in skin condition, including flattening of the lesions and
a decrease in scaling. Shortly after the second administration of J695 (5
mg/kg IV), the subject's skin was totally cleared of psoriatic lesions,
in the absence of any local treatment. Erythematous plaques covered with
white scales reappeared concomitant with the expected clearance of J695
after the second administration of antibody.

Example 10

Comparison of J695 Produced by Two CHO Cell Lines

[0785] For recombinant expression of J695, a recombinant expression vector
encoding both the antibody heavy chain and the antibody light chain is
introduced into dhfr-CHO cells (Urlaub, G. and Chasin, L. A. (1980) Proc.
Natl. Acad. Sci. USA 77:4216-4220) by calcium phosphate-mediated
transfection. Within the recombinant expression vector, the antibody
heavy and light chain genes are each operatively linked to
enhancer/promoter regulatory elements (e.g., derived from SV40, CMV,
adenovirus and the like, such as a CMV enhancer/AdMLP promoter regulatory
element or an SV40 enhancer/AdMLP promoter regulatory element) to drive
high levels of transcription of the genes. The recombinant expression
vector also carries a DHFR gene, which allows for selection of CHO cells
that have been transfected with the vector using methotrexate
selection/amplification.

[0786] One hundred and fifty micrograms of an expression vector encoding
the peptide sequences of the human antibody J695 were dissolved in 2.7 ml
water in a 50 ml conical tube. Three hundred μL of 2.5 M CaCl2
were added and this DNA mixture was added dropwise to 3 ml of
2×HEPES buffered saline in a 50 ml conical tube. After vortexing
for 5 sec and incubating at room temperature for 20 min, 1 mL was
distributed evenly over each plate (still in F12 medium), and the plates
were incubated at 37° C. for 4 h. Liquid was removed by aspiration
and 2 ml of 10% DMSO in F12 were added to each plate. The DMSO shock
continued for 1 min, after which the DMSO was diluted by the addition of
5 ml PBS to each plate. Plates were washed twice in PBS, followed by the
addition of 10 ml of alpha MEM, supplemented with H/T and 5% FBS
(selective for cells expressing DHFR) and overnight incubation at
37° C. Cells were seeded into 96-well plates at a density of 100
cells per well, and plates were incubated at 37° C., 5% CO2
for two weeks, with one change of medium per week.

[0787] Five days after the final medium change, culture supernatants were
diluted 1:50 and tested using an ELISA specific for human IgG gamma
chain. The clones yielding the highest ELISA signal were transferred from
the 96-well plates to 12-well plates in 1.5 ml/well of Alpha MEM+5%
dialyzed serum. After 3 days, another ELISA specific for human IgG gamma
chain was performed, and the 12 clones with the greatest activity were
split into the alpha MEM+5% dialyzed serum and 20 nM MTX. Cell line
031898 218 grew in the presence of 20 nM MTX without any apparent cell
death or reduction in growth rate, produced 1.8 μg/ml hIgG in a
three-day assay. T-25 cultures of 031898 218, growing in medium
containing MTX, produced an average of 11.9 μg/ml of J695. The line,
designated ALP903, was adapted to growth in suspension under serum-free
conditions, where it produced 7.5 pg J695/cell/24 h.

[0788] ALP903 cells, after initial selection in alpha MEM/5% FBS/20 nM MTX
medium, were passed again in 20 nM MTX. The cells were cultured under 100
nM MTX selection, followed by passaging in 500 nM MTX twice in the next
30 days. At that time the culture was producing 32 μg J695/mL/24 h.
The culture was subcloned by limiting dilution. Subclone 218-22 produced
16.5 μg/mL in a 96-well plate in 2 days and 50.3 μg/mL of J695 in a
12-well dish in 2 days. Clone 218-22 was cultured in alpha MEM/5%
dialyzed FBS/500 nM MTX for 38 days, followed by adaptation to serum-free
spinner culture, as above. The average cell-specific productivity of the
serum-free suspension culture, designated ALP 905, was 58 pg/cell/24 h.

[0789] The first cell line used to produce J695 (ALP 903) resulted in
lower yields of the antibody from culture than a second cell line, ALP
905. To assure that the ALP 905-produced J695 was functionally identical
to that produced from ALP 903, both batches of antibodies were assessed
for IL-12 affinity, for the ability to block IL-12 binding to cellular
receptors, for the ability to inhibit IFN-γ induction by IL-12, and
for the ability to inhibit IL-12-mediated PHA blast proliferation.

[0790] The affinities of J695 batches ALP 903 and ALP 905 for IL-12 were
determined by measuring the kinetic rate constants of binding to IL-12 by
surface plasmon resonance studies (BIAcore analyses). The off-rate
constant (kd) and the on-rate constant (ka) of antibody batches
ALP903 and ALP905 for binding to rhIL-12 were determined in three
experiments (as described in Example 3). The affinity, Kd, of
binding to IL-12 was calculated by dividing the off-rate constant by the
on-rate constant. Kd was calculated for each separate experiment and
then averaged. The results showed that the determined kinetic parameters
and affinity of binding to rhIL-12 were very similar for J695 batches ALP
903 and ALP 905: the calculated Kd was 1.19±0.22×10-10
M for batch ALP 903 and 1.49±0.47×10-10 M for batch ALP 905
(see Table 13).

[0791] The ability of J695 derived from both ALP 903 and ALP 905 to block
binding of rhIL-12 to IL-12 receptors on human PHA-activated
T-lymphoblasts was assessed (see Example 3). Each sample of J695 was
tested at a starting concentration of 1×10-8 with 10-fold
serial dilutions. The antibody was preincubated for 1 hour at 37°
C. with 50 pM [125I]-human IL-12 in binding buffer. PHA blast cells
were added to the antibody/[125I]-human IL-12 mixture and incubated
for 2 h at room temperature. Cell bound radioactivity was separated from
free [125I]-IL-12 by centrifugation and washing steps, and %
inhibition was calculated. The IC50 values for J695 were determined
from the inhibition curves using 4-parameter curve fitting and were
confirmed by two independent experiments. Incubations were carried out in
duplicate. The results for the two batches of J695 were very similar (see
Table 13).

[0792] The ability of J695 from both ALP 903 and ALP 905 cells to inhibit
rhIL-12-induced IFN-γ production by human PHA-activated
lymphoblasts in vitro was assessed. Serial dilutions of J695 were
preincubated with 200 pg/mL rhIL-12 for 1 h at 37° C. PHA
lymphoblast cells were added and incubated for 18 hours at 37° C.
After incubation, cell free supernatant was withdrawn and the level of
human IFN-γ determined by ELISA. The IC50 values from the
inhibition curves were plotted against the antibody concentration using
4-parameter curve fitting. The results demonstrate that the ability of
the two batches to inhibit IFN-γ production is very similar.

[0793] The in vitro PHA blast cell proliferation assay was used to measure
the neutralization capacity of ALP 903 and ALP 905 J695 for rhIL-12.
Serial dilutions of J695 of each type were preincubated with 230 pg/mL
human IL-12 for 1 h at 37° C. Next PHA blast cells were added and
incubated for 3 days at 37° C. The cells were then labeled for 6
hours with 1 γCi/well [3H]-thymidine. The cultures were
harvested and [3H]-thymidine incorporation measured. Non-specific
proliferation (background) was measured in the absence of rhIL-12. The
IC50 values for ALP 903 and ALP 905 J695 were found to be very
similar and are set forth in Table 13.

[0795] Real-time biospecific interaction analysis (BIA) based on surface
plasmon resonance technology was used to map the epitope specificity
patterns of J695 and 4 other monoclonal antibodies against soluble
recombinant human IL12. The technique does not require labeling of either
antibodies or antigen. Antibodies directed against separate and distinct
epitopes will bind simultaneously to the antigen, whereas antibodies
directed against closely related epitopes will interfere with each
other's binding. Furthermore, if the second antibody fails to bind, the
epitopes defined by the two antibodies may be identical or overlapping,
or binding of the first antibody may prevent binding of the second
antibody through allosteric inhibition caused by a conformational
alteration in the target molecule.

[0796] An epitope mapping assay using Biacore was performed. First,
carboxyl groups on the dextran matrix were activated with 100 mM
N-hydroxysuccinimide (NHS) and 400 mM
N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC),
across four different flow cells. Next, Antibody 1 was injected across
the activated matrix. Approximately fifty microliters of anti-rhIL12
antibody (25 μg/ml), diluted in sodium acetate, pH 4.5, was injected
across the activated biosensor and free amines on the protein are bound
directly to the activated carboxyl groups. Typically, 5000 resonance
units were immobilized. Unreacted matrix EDC-esters were deactivated by
an injection of 1 M ethanolamine. Standard amine coupling kits were
commercially available (Biacore AB, Cat. No. BR-1000-50, Uppsala,
Sweden). SPR measurements were performed using CM biosensor chip (Biacore
AB, Cat No BR-1000-14, Uppsala, Sweden). All antibodies and antigens to
be analyzed on the biosensor surface were diluted in HBS-EP running
buffer (Biacore AB, Cat No BR-1001-88, Uppsala, Sweden).

[0797] Next, rhuIL12 (100 nM) was injected across covalently immobilized
antibody on the CM5 biosensor surface at a flow rate of 25 μl/min.
Before injection of the antigen and immediately afterward, HBS-EP buffer
alone flowed through each flow cell. Excess soluble Antibody 2 (25
μg/ml) was then injected across captured rhuIL12 (5 minute contact
time). Before injection of Antibody 2 and immediately afterward, HBS-EP
buffer alone flowed through each flow cell. The net difference in the
signals between the baseline and the point corresponding to approximately
30 seconds after completion of Mab injection was taken to represent the
final binding value. Again, the response was measured in Resonance Units.
Biosensor matrices were regenerated using 10 mM HCl (5 minute contact
time) before injection of the next sample.

[0798] Antibodies and antigens supplied by Abbott Bioresearch Center
and/or commercial vendors were as follows: Human J695; Mouse C8.6.2 Mab;
Human 1D4.7 Mab; Human C340 Mab; Mouse 7G3 Mab; Human IgG1 control (1.0
mg/ml, Sigma Catalog No. 1-3889). Each of the antibodies used in this
study binds to an epitope of the p40 subunit of human IL-12. The
antibodies ID4.7 and 7G3 are described in U.S. Provisional Patent
Application No. 60/695,679, filed Jun. 29, 2005, and U.S. patent
application Ser. No. 11/478,096, filed Jun. 29, 2006. The antibody C340
is described in U.S. Pat. No. 7,063,964 and U.S. Pat. No. 6,902,734. The
entire contents of each of the foregoing patents and patent applications
are hereby incorporated herein by reference. The antibody C8.6.2 is a
subclone of, and thus has the same characteristics as, the antibody C8.6,
which is described in A. D'Andrea et al., 1992 J. Exp. Med.
176:1387-1398, the entire contents of which are hereby incorporated
herein by reference. Recombinant human interleukin 12 (rhIL12,
commercially available by Wyeth).

[0800] The results described in this Example demonstrate that Biacore can
be used to characterize monoclonal antibody epitopes. Each antibody and
antigen was injected over a long time period, until a plateau was reached
in the SPR signal. Examination of the sensorgrams showed that all human
anti-rhIL12 monoclonal antibodies bound specifically to rhIL12 when they
were directly immobilized across carboxymethyl dextran surface. The
results presented in this Example demonstrate that the antibodies that
can bind simultaneously to IL-12 are binding to non-overlapping epitopes.
In particular, 7G3 binds to a different epitope that is not shared by
J695, C8.6.2, C340 or 1D4.7. The results further demonstrate that the
antibodies tested that cannot bind simultaneously to IL-12 are binding to
overlapping epitopes (i.e., no binding of antibody 2 observed when IL-12
is bound to antibody 1). In particular, C8.6.2, C340 and 1D4.7 bind to
overlapping epitopes as binding of one of these antibodies to IL-12 does
not allow binding of the second antibody in all cases. Finally, the
results demonstrate that the antibodies that cannot bind simultaneously
to IL-12 are prevented from binding due to an allosteric interaction that
prevents the antibodies from binding simultaneously (i.e., antibody 2
releases IL-12 from antibody 1). In particular, J695 cannot bind rhuIL-12
simultaneously with either C8.6.2, C340 or 1D4.7. The inability of the
antibodies to bind IL-12 simultaneously does not indicate a simple
competition between the antibodies for the same binding site, but rather
that a conformational alteration in the target molecule IL-12 occurs upon
binding. This unusual allosteric mechanism is evident in the observation
that soluble J695 rapidly displaced IL-12 bound to immobilized C8.6.2,
C340 or 1D4.7. Conversely, C8.6.2, C340 and 1D4.7 slowly displaced IL-12
bound to immobilized J695.

Example 12

Identification of Antibodies Capable of Altering the Conformational
Structure of an Interleukin Containing a p40 Subunit

[0801] Antibodies are identified that bind to the p40 subunit of an
interleukin, e.g., IL-12 or IL-23, and are capable of altering its
conformational structure by using real-time biospecific interaction
analysis (BIA). The experimental protocol used is essentially as
described above in Example 11.

[0802] Specifically, carboxyl groups on a dextran matrix are activated,
typically with 100 mM N-hydroxysuccinimide (NHS) and 400 mM
N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).
Next, a first antibody that specifically binds to the p40 subunit of an
interleukin, e.g., IL-12 or IL-23, is then injected across the activated
matrix. In particular, the first antibody injected across the activated
matrix is an antibody that binds to an epitope of the p40 subunit of
IL-12 to which the antibodies C8.6.2, C340 or 1D4.7 bind. Preferably, the
first antibody is either C8.6.2, C340 or 1D4.7. Approximately fifty
microliters of the first antibody (e.g., at 25 μg/ml, diluted in
sodium acetate, pH 4.5) is then injected across the activated biosensor
and free amines on the protein are bound directly to the activated
carboxyl groups. Typically, 5000 resonance units are immobilized.
Unreacted matrix EDC-esters are deactivated by an injection of 1 M
ethanolamine. SPR measurements are performed using CM biosensor chip
(Biacore AB, Cat No BR-1000-14, Uppsala, Sweden) as described above in
Example 11. All antibodies and antigens to be analyzed on the biosensor
surface are diluted in HBS-EP running buffer (Biacore AB, Cat No
BR-1001-88, Uppsala, Sweden).

[0803] Next, the p40 subunit of the interleukin, e.g., IL-12 or IL-23
(e.g., 100 nM) is injected across the covalently immobilized antibody on
the CM5 biosensor surface, typically at a flow rate of approximately 25
μl/min. The p40 subunit of the interleukin, e.g., IL-12 or IL-23, can
be injected, for example, either as an isolated single subunit (or
fragment thereof) or alternatively can be injected in a heterodimeric
form, wherein the heterodimer comprises the p40 subunit and a second
subunit, e.g., the heterodimer p40/p35 (IL-12), or an alternate
heterodimer comprising the p40 subunit of IL-12 and a p19 subunit
(p40/p19; IL-23). Before injection of the antigen and immediately
afterward, HBS-EP buffer alone is flowed through each flow cell.
Following this, an excess of a soluble test antibody (e.g., at a
concentration of 25 μg/ml) is injected across the captured p40 subunit
of the interleukin (e.g., IL-12 or IL-23), typically for a contact time
of approximately 5 minutes. Before injection of the test antibody and
immediately afterward, HBS-EP buffer alone is again flowed through each
flow cell. The net difference in the signals between the baseline and the
point corresponding to approximately 30 seconds after completion of
antibody injection is taken to represent the final binding value and is
measured in Resonance Units. Antibodies which rapidly displace the p40
subunit of the interleukin (e.g., IL-12 or IL-23) from the first antibody
immobilized on the CM5 biosensor surface are identified as antibodies
that can alter the conformational structure of the interleukin, e.g., the
p40 subunit of the interleukin.

[0804] This conformational alteration in the structure of the interleukin
((e.g., IL-12 or IL-23) may be confirmed by any of the well known
techniques in the art for monitoring 3-dimensional structures of
proteins. For example X-ray crystallography or Circular Dichroism may be
used.

EQUIVALENTS

[0805] Those skilled in the art will recognize, or be able to ascertain
using no more than routine experimentation, many equivalents to the
specific embodiments of the invention described herein. Such equivalents
are intended to be encompassed by the following claims

Sequence CWU
0

SEQUENCE LISTING
<160> NUMBER OF SEQ ID NOS: 675
<210> SEQ ID NO 1
<211> LENGTH: 6
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 could be either His or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 4 could be either Tyr or
His
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 6 could be either Tyr, Asn
or
Thr
<400> SEQUENCE: 1
Xaa Gly Ser Xaa Asp Xaa
1 5
<210> SEQ ID NO 2
<211> LENGTH: 12
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 2 could be either Ser or
Thr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 4 could be either Asp or
Glu
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 5 could be either Ser, Arg
or
Lys
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 6 could be either Ser, Gly
or
Tyr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 7 could be either Leu, Phe,
Thr
or Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 8 could be either Arg, Ser,
Thr, Trp or His
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 9 could be either Gly or
Pro
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 10 could be either Ser,
Thr,
Ala or Leu
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 11 could be either Arg,
Ser,
Met, Thr or Leu
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 12 could be either Val,
Ile,
Thr, Met or Leu
<400> SEQUENCE: 2
Gln Xaa Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
<210> SEQ ID NO 3
<211> LENGTH: 17
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<400> SEQUENCE: 3
Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> SEQ ID NO 4
<211> LENGTH: 7
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 could be either Gly or
Tyr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 3 could be either Asp or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 4 could be either Gln or
Asn
<400> SEQUENCE: 4
Xaa Asn Xaa Xaa Arg Pro Ser
1 5
<210> SEQ ID NO 5
<211> LENGTH: 9
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa represents either Ser or Glu
<400> SEQUENCE: 5
Phe Thr Phe Ser Xaa Tyr Gly Met His
1 5
<210> SEQ ID NO 6
<211> LENGTH: 13
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 could be either Ser or
Thr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 3 could be either Ser or
Gly
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 4 could be either Arg or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 8 could be either Gly or
Val
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 9 could be either Ser or
Ala
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 10 could be either Asn, Gly
or
Tyr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 11 could be either Thr or
Asp
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 13 could be either Lys or
His
<400> SEQUENCE: 6
Xaa Gly Xaa Xaa Ser Asn Ile Xaa Xaa Xaa Xaa Val Xaa
1 5 10
<210> SEQ ID NO 7
<211> LENGTH: 115
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 6 could be either Gln or
Glu
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 16 could be either Arg or
Gly
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 31 could be either Ser or
Glu
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 84 could be either Lys or
Asn
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 97 could be either Thr, Ala
or
Lys
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 98 could be either Thr or
Lys
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 99 could be either Ser or
His
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 102 could be either Tyr or
His
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 104 could be either Tyr,
Asn or
Thr
<400> SEQUENCE: 7
Gln Val Gln Leu Val Xaa Ser Gly Gly Gly Val Val Gln Pro Gly Xaa
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Asx
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Xaa Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Xaa Xaa Xaa Gly Ser Xaa Asp Xaa Trp Gly Gln Gly Thr Met Val Thr
100 105 110
Val Ser Ser
115
<210> SEQ ID NO 8
<211> LENGTH: 112
<212> TYPE: PRT
<213> ORGANISM: Homo sapiens
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 1 could be either Ser or
Gln
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 2 could be either Tyr or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 13 could be either Thr or
Ala
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 23 and 91 could be either
Ser
or Thr
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 25 could be either Gly or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 26 could be either Arg or
Ser
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 30 could be either Gly or
Val
<220> FEATURE:
<223> OTHER INFORMATION: Xaa at position 31 could be either Ser or
Ala
<220> FEATURE: